Method of uncoupling the catabolic pathway of glycolysis from the oxidative membrane bound pathway of glucose conversion

ABSTRACT

The invention provides methods for producing products comprising improved host cells genetically engineered to have uncoupled productive and catabolic pathways. In particular, the present invention provides host cells having a modification in nucleic acid encoding an endogenous enzymatic activity that phosphorylates D-glucose at its 6th carbon and/or a modification of nucleic acid encoding an enzymatic activity that phosphorylates D-gluconate at its 6th carbon. Such improved host cells are used for the production of products, such as, ascorbic acid intermediates. Methods for making and using the improved host cells are provided. Nucleic acid and amino acid sequences for glucokinase and gluconokinase are provided.

The present application claims priority to U.S. Provisional PatentApplication Ser. No. 60/281,618 filed Apr. 4, 2001 and U.S. ProvisionalApplication Ser. No. 60/282,259 filed Apr. 5, 2201.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with the United States Government support underAward No. 70 NANB 5H1138 awarded by the United States Department ofCommerce. The Government has certain rights in this invention.

TECHNICAL FIELD

The present invention relates to engineering of metabolic pathways ofhost cells and provides methods and systems for the production ofproducts in host cells. In particular, the invention provides methodsfor producing products in host cells which have been geneticallyengineered to have uncoupled productive and catabolic pathways.

BACKGROUND ART

In the initial stage of host cell carbohydrate metabolism, that is,glycolysis, each glucose molecule is converted to two molecules ofpyruvate in the cytosol. The chemical reactions that convert glucose topyruvate are referred to as the Embden-Meyerhoff pathway. All of themetabolic intermediates between the initial carbohydrate and the finalproduct, pyruvate, are phosphorylated compounds. The final stage ofoxidation of carbohydrates, the citric acid cycle, is a complex set ofreactions that also takes place in the cytosol. The reactions in theEmbden-Meyerhoff pathway and citric acid cycle result in the conversionof carbohydrate molecules to CO₂ molecules with the concomitantreduction of NAD+ to NADH molecules and the formation of ATP.

The central metabolic routes produce NADH or NADPH. In general NADPH isutilized in biosynthetic reactions and NADH is rapidly reoxidized in twoways:

-   -   (1) In fermentative pathways by the direct reduction of organic        metabolites.    -   (2) In respiratory processes by electron transport through a        respiratory chain to a terminal electron acceptor. This acceptor        is usually O₂, but in some cases can be productive ions,        including nitrate and sulfate. In all respiratory processes, ATP        is generated.

Some bacteria posses the ability to oxidize some substratesextracellularly, producing useful oxidation products such as L-sorbose,D-gluconate, keto-gluconates, etc. Such oxidation reactions are calledproductive fermentation since they involve incomplete substrateoxidation, accompanying accumulation of corresponding oxidation productin large amounts in the growth medium. The oxidation reaction is coupledto the respiratory chain of the microorganism. (Bacterial Metabolism2^(nd) Edition (1985) Springer-Verlag, New York, N.Y.).

Bacteria which ferment glucose through the Embden-Meyerhof pathway, suchas members of Enterobacteriacea and Vibrionaceae, are described inBouvet, et al. (1989) International Journal of Systematic Bacteriology,39:61-67. Pathways for metabolism of ketoaldonic acids in Erwinia sp.are described in Truesdell, et al, (1991) Journal of Bacteriology,173:6651-6656.

Host cells having mutations in enzymes involved in glycolysis have beendescribed. Yeast having mutations in glucokinase are described inHarrod, et al. (1997) J. Ind. Microbiol. Biotechnol. 18:379-383;Wedlock, et al. (1989) J. Gen. Microbiol. 135: 2013-2018; and Walsh etal. (1983) J. Bacteriol. 154:1002-1004. Bacteria deficient inglucokinase have been described. Pediococcus sp. deficient inglucokinase is described in Japanese patent publication JP 4267860.Bacillus sphaericus lacking glucokinase is described in Russell et al.(1989) Appl. Environ. Microbiol. 55: 294-297. Penicillium chrysogenumdeficient in glucokinase is described in Barredo et al.(1988)Antimicrob. Agents-Chemother 32: 1061-1067. A glucokinase-deficientmutant of Zymomonas mobilis is described in DiMarco et al. (1985) Appl.Environ. Microbiol. 49:151-157.

Many bacteria posses an active transport system known asPhosphotransferase transport System (PTS) that couples the transport ofa carbon source to its phosphorylation. In this system, the phosphorylgroup is transferred sequentially from phosphoenolpyruvate (PEP) toenzyme I and from enzyme I to protein HPr. The actual translocation stepis catalyzed by a family of membrane bound enzymes (called enzyme II),each of which is specific for one or a few carbon sources. Consideringthat PTS consumes PEP to phosphorylate the carbon source, and PEP is acentral metabolite used in for many biosynthetic reactions, it maydecrease the efficiency of conversion of a carbon source into a desiredproduct. this transport system has been replaced by a permease andglucokinase from an heterologous origin as described by Parker et al.(1995) Mol. Microbiol. 15: 795-802. or homologous origin as reported byFlores et al. (1996) Nat. Biotechnol. 14: 620-623. In both of these 2examples, the function of the PTS system for glucose transport andphosphorylation was replaced by a glucose permease and a glucokinaseactivities.

Products of commercial interest that have been produced biocatalyticallyin genetically engineered host cells include intermediates of L-ascorbicacid; 1,3-propanediol; glycerol; D-gluconic acid; aromatic amino acids;3-deozy-D-arabino-heptulosonate 7-phosphate (DAHP); and catechol, amongothers.

L-Ascorbic acid (vitamin C, ASA) finds use in the pharmaceutical andfood industry as a vitamin and antioxidant. The synthesis of ASA hasreceived considerable attention over many years due to its relativelylarge market volume and high value as a specialty chemical.

The Reichstein-Grussner method, a chemical synthesis route from glucoseto ASA, was first disclosed in 1934 (Helv. Chim. Acta 17:311-328).Lazarus et al. (1989, “Vitamin C: Bioconversion via a Recombinant DNAApproach”, Genetics and Molecular Biology of Industrial Microorganisms,American Society for Microbiology, Washington D.C. Edited by C. L.Hershberger) disclose a bioconversion method for production of anintermediate of ASA, 2-keto-L-gulonic acid (2-KLG, KLG) which can bechemically converted to ASA. This bioconversion of carbon source to KLGinvolves a variety of intermediates, the enzymatic process beingassociated with co-factor dependent 2,5-DKG reductase activity (2,5-DKGRor DKGR).

Many bacterial species have been found to contain DKGR, particularlymembers of the Coryneform group, including the genera Corynebacterium,Brevibacterium, and Arthrobacter. DKGR obtained from Corynebacterium sp.strain SHS752001 is described in Grindley et al. (1988, Applied andEnvironmental Microbiology 54:1770-1775). DKGR from Erwinia herbicola isdisclosed in U.S. Pat. No. 5,008,193 to Anderson et al. Other reductasesare disclosed in U.S. Pat. Nos. 5,795,761; 5,376,544; 5,583,025;4,757,012; 4,758,514; 5,004,690; and 5,032,514.

1,3-Propanediol is an intermediate in the production of polyester fibersand the manufacture of polyurethane and cyclic compounds. The productionof 1,3-propanediol is described in U.S. Pat. Nos. 6,025,184 and5,686,286. 1,3-propanediol can be produced by the fermentation ofglycerol. The production of glycerol is described in TWO 99/28480 andTWO 98/21340.

D-gluconic acid and its derivatives have been used commercially asagents in textile bleaching and detergents. The production of D-gluconicacid in Bacillus species lacking gluconokinase activity and having highglucose dehydrogenase activity is described in TWO 92/18637.

The production of members of the aspartate family of amino acids isdescribed in U.S. Pat. No. 5,939,307. The production of riboflavin(Vitamin B2) is described in TWO 99/61623.

Many cyclic and aromatic metabolites are derived from DHAP includingtyrosine, tryptophan and phenylalanine. The production of DAHP isdescribed in U.S. Pat. No. 5,985,617. Catechol is a starting materialfor the synthesis of pharmaceuticals, pesticides, flavors, fragrancesand polymerization inhibitors. The production of catechol is describedin U.S. Pat. No. 5,272,073.

However, there are still problems associated with these productionmethodologies. One such problem is the diversion of carbon substratesfrom the desired productive pathways to the catabolic pathways. Suchdiversion results in the loss of available carbon substrate material forconversion to the desired productive pathway products and resultantenergy costs, ATP or NADPH, associated with the transport orphosphorylation of the substrate for catabolic pathway use.

In spite of the advances made in the production of products by hostcells, there remains a need for improved host cells for use in theproduction of desired products. The present invention addresses thatneed.

SUMMARY

Methods for the production of products in recombinant host cellsgenetically engineered to have uncoupled productive and catabolicpathways during part or all of the production are provided. The presentinvention also provides recombinant host cells genetically engineered tocomprise productive and/or catabolic pathways that are uncoupled or thatcan be regulated during production, and methods for their preparation.

Accordingly, the invention provides a process for producing a product ina recombinant host cell comprising, culturing a host cell capable ofproducing said product in the presence of a carbon source underconditions suitable for the production of said product wherein said hostcell comprises productive and catabolic pathways, wherein said pathwaysare uncoupled during part or all of said culturing. In some embodiments,the productive pathway and catabolic pathway are uncoupled during all ofsaid culturing. In some embodiments, the product being produced is acomponent of the productive pathway or the host cell. In otherembodiments, the product being produced is a component of the catabolicpathway of the host cell. In further embodiments, the product beingproduced is encoded by nucleic acid recombinantly introduced into thehost cell.

In some embodiments, the productive pathway is in the host cellmembrane. In other embodiments, the catabolic pathway is intracellular.In further embodiments, the productive pathway and catabolic pathway areuncoupled at the stage of initial phosphorylation of said carbon source.In additional embodiments, the productive pathway and catabolic pathwayare uncoupled at the stage of phosphorylation of a carbon metabolite.

In further embodiments, the uncoupling of the productive pathway andcatabolic pathway comprises inhibition of at least one enzymaticactivity that phosphorylates a carbon source and/or a carbon metaboliteduring said culturing. In other embodiments, the uncoupling of saidproductive pathway and said catabolic pathway comprises inactivation ofat least one enzymatic activity that phosphorylates said carbon sourceand/or a carbon metabolite during part or all of said culturing.

In additional embodiments, the host cell comprises a mutation in ordeletion of part or all of a polynucleotide that encodes an enzymaticactivity that couples an productive pathway with a catabolic pathway. Inyet other embodiments, the host cell comprises at least onepolynucleotide that lacks the encoding for an enzymatic activity thatphosphorylates said carbon source and/or a carbon metabolite whereinsaid polynucleotide is operably linked to a regulatable promoter.

In some embodiments, the enzymatic activities that are reduced orinactivated are those that phosphorylate D-glucose at its 6th position.In other embodiments, the enzymatic activity that is reduced orinactivated phosphorylates D-gluconic acid at its 6th position. Infurther embodiments, the enzymatic activity that phosphorylatesD-glucose at its 6th carbon includes glucokinase, phosphoenol pyruvatesynthase (PEP) or phosphotransferase system (PTS). In additionalembodiments, the enzymatic activity that phosphorylates D-gluconate atits 6th carbon includes gluconokinase.

In some embodiments, the product is recovering and in other embodiments,the product is converted into a second product. The host cell includesGram negative or Gram positive host cells. In some embodiments, the hostcell is an Enterobacteriaceae host cell that includes Erwinia,Enterobacter, Gluconobacter, Acetobacter, Coyrnebacteria, Escherchia orPantoea. In other embodiments, the host cell is an that includesBacillus and Pseudomonas.

In other embodiments, the host cell can be any bacteria that naturallyor after proper genetic modifications, is able to utilize one carbonsource to maintain certain cell functions, for example, but not limitedto, the generation of reducing power in the form of NAD, FADH₂ or NADPH,while another carbon source is converted into one or more product(s) ofcommercial interest.

In some embodiments, the uncoupling of the productive and catabolicpathways allows the production of compounds generally derived from thecatabolic pathway, wherein those products generally derived from theproductive pathways are utilized to satisfy the metabolic demands of thehost cell. In other embodiments, the uncoupling of the productive andcatabolic pathway allows the production of compounds generally derivedfrom the productive pathway, whereas those products derived fromcompounds present in the catabolic pathway satisfy the metabolic demandsof the host cell.

In some embodiments, the product includes those products generallyderived from the catabolic pathway include those derived from fructose,the pentose pathway and the TCA cycle. In other embodiments, the productincludes those generally derived from the productive pathway, e.g., anascorbic acid intermediate including GA, KDG, DKG, KLG or IA.

The invention also provides host cells comprising an productive pathwayand a catabolic pathway, wherein said productive pathway and saidcatabolic pathways are uncoupled. In some embodiments, the host cellscomprise a modification of the polynucleotide encoding an enzymaticactivity such that such enzymatic activity is reduced or inactivated.One such modification precludes the host cell from phosphorylatingD-glucose at it 6th carbon and/or precludes a host cell fromphosphorylating D-gluconic acid at its 6th carbon, wherein one or bothof said polynucleotides is modified. In some embodiments, the enzymaticpathway that is inactivated includes that of hexokinase, glucokinase;gluconokinase; phosphoenol pyruvate synthase (PEP); orphosphotransferase system (PTS).

The present invention also provides methods for producing host cellshaving modified levels of enzymatic activities. The present inventionalso provides novel nucleic acid and amino acid sequences for which lackenzymatic activity that phosphorylates D-glucose at its 6th carbon andenzymatic activity that phosphorylates D-gluconate at its 6th carbon.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic representation of some of the metabolicroutes involved in Glucose assimilation in Pantoea citrea. The enzymaticsteps affected by the genetic modifications described in the presentinvention, are indicated by an X. Boxes labeled with a T representputative transporters.

FIG. 2. Some possible catabolic routes that can be used to channelglucose into cellular metabolism. The arrows represent at least oneenzymatic step.

FIG. 3. depicts products that can be obtained from indicated commercialroutes. The majority of the carbon used to synthesize the compoundslisted on the left side, can be obtained from the catabolic pathway orTCA cycle. On the contrary, the compounds on the right, derive most ofits carbon from the pentose pathway and/or from the oxidation of glucoseinto keto-acids.

FIG. 4 depicts a nucleic acid (SEQ ID NO:1) for a Pantoea citreaglucokinase

FIG. 5 depicts an amino acid (SEQ ID NO:2) sequence for a Pantoea citreaglucokinase.

FIG. 6 depicts a nucleic acid (SEQ ID NO:3) for a Pantoea citreagluconokinase

FIG. 7 depicts an amino acid (SEQ ID NO:4) sequence for a Pantoea citreagluconokinase.

FIG. 8 depicts amino acid (SEQ ID NO: 5-10) for the genes glk 30, glk31, gnt 1, gnt 2, pcgnt 3 and pcgnt 4.

FIG. 9 depicts D-glucose, D-gluconate and some of their derivatives. Thestandard numbering of the carbons on glucose is indicated by the numbers1 and 6. 2-KDG=2-keto-D-gluconate; 2,5-DKG=2,5-diketogluconate;2KLG=2-keto-L-gulonate.

FIG. 10 depicts general strategy used to interrupt the gluconate kinasegene from P. citrea.

FIG. 11 depicts the oxidative pathway for the production of ascorbicacid. E1 stands for glucose dehydrogenase; E2 stands for gluconic aciddehydrogenase; E3 stands for 2-keto-D-gluconic acid dehydrogenase; andE4 stands for 2,5-diketo-D-gluconic acid reductase.

FIG. 12 depicts the net reactions during the fermentation of host cellscapable of producing ascorbic acid intermediates.

FIG. 13 depicts carbon evolution rate (CER) and oxygen uptake rate (OUR)of a fermentation of a wild-type organism after exposure to glucose.

FIG. 14 depicts the CER and OUR of a fermentation with a single delete(glucokinase).

FIG. 15 depicts the CER and OUR of a fermentation with a single delete(gluconokinase).

FIG. 16 depicts the CER and OUR of a fermentation with a host cellhaving both glucokinase and gluconokinase deleted.

FIG. 17 is a schematic illustrating the interrelationships of variousmetabolic pathways (including the glycolytic pathway, TCA cycle, andpentose pathway) and the oxidative pathways. Glk=glucokinase;Gntk=gluconokinase; IdnO=5-keto-D-gluconate 5-reductase; IdnD=L-ldonate5-dehydrogenase; TKT=transketolase; TAL=transaldolase, 2KR=2-ketoreductase; 2,5DKGR=2,5-diketogluconate reductase.

FIG. 18 is a schematic illustrating the interrelationships of variouscentral metabolic pathways and the modifications which would increasethe production of ribose. The X indicate the enzymatic steps that wouldbe modified to effect the desired increase in ribose yield.

FIG. 19 is a schematic illustrating the interrelationships of variouscentral metabolic pathways and the modifications which would increasethe production of riboflavin. The X indicate the enzymatic steps thatwould be modified to effect the desired increase in ribose yield.

FIG. 20 is a schematic illustrating the interrelationships of variouscentral metabolic pathways and the modifications which would increasethe production of nucleotides. The X indicate the enzymatic steps thatwould be modified to effect the desired increase in nucleotide yield.

FIG. 21 is a schematic illustrating the interrelationships of variouscentral metabolic pathways and the modifications which would increasethe production of tartrate. The X indicate the enzymatic steps thatwould be modified to effect the desired increase in ribose production.IdnO=5-keto-D-gluconate 5-reductase;

IdnD=I-Idonate 5-dehydrogenase.

FIG. 22 is a schematic illustrating the interrelationships of variouscentral metabolic pathways and the modifications which would increasethe production of gluconateribose. The X indicate the enzymatic stepsthat would be modified to effect the desired increase in gluconateproduction.

FIG. 23 is a schematic illustrating the interrelationships of variouscentral metabolic pathways and the modifications which would increasethe production of erythorbic acid. The X indicate the enzymatic stepsthat would be modified to effect the desired increase in erythorbic acidproduction.

FIG. 24 is a schematic illustrating the interrelationships of variouscentral metabolic pathways and the modifications which would increasethe production of 2,5-DKG. The X indicate the enzymatic or transportpathways that would be modified to effect the desired increase in2,5-diketogluconate production.

FIG. 25 is a schematic illustrating the pathway of dihydroxyacetonephosphate (DHAP) being converted to glycerol.

FIG. 26 depicts the DNA Sequence of primers used to amplify by PCR the2.9 kb DNA fragment that contains the glpK gene as described in Example7.

FIG. 27 describes the DNA sequence of the structural gene of theglycerol kinase from P. citrea as described in Example 7. The sequenceof the Hpal sited used to interrupt the gensis underlined.

FIG. 28 depicts the protein sequence of the glycerol kinase from P.citrea as described in Example 7.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for producing products comprisingrecombinant host cells that comprise an productive pathway and acatabolic pathway wherein said pathways are uncoupled in the host cellduring part or all of said method, that is wherein said pathways do notcompete for initial carbon source, such as D-glucose or D-gluconic acidfor example, or for cellular components, such as co-factor and ATPduring part or all of said method. The invention encompasses methodswherein the productive and catabolic pathways are uncoupled viamodification and/or regulation of enzymatic activities present in theproductive and/or catabolic pathways.

The invention encompasses methods wherein the productive and catabolicpathway are coupled during part of said culturing, for example duringthe early part of said culturing where it is desirable to channel ordirect host cell resources to building host cell biomass, and uncoupledduring part of said culturing, for example, after host cell biomass hasbeen produced or when it is desirable to channel or direct cellresources to production of product. The invention encompasses methodscomprising culturing recombinant host cells having a productive pathwayand a catabolic pathway that are uncoupled during all of said culturing.

The invention encompasses methods wherein the productive and catabolicpathways are uncoupled at the stage of initial phosphorylation of thecarbon source that is used by the cell, by modifying the genomicsequence that encodes such phosphorylation.

The uncoupling of the productive pathway and catabolic pathwayencompasses inhibition of at least one enzymatic activity thatphosphorylates the initial carbon source and/or any carbon metabolite inthe productive and/or catabolic pathway. The uncoupling of theproductive pathway and catabolic pathway encompasses inactivation of atleast one enzymatic activity that phosphorylates the initial carbonsource and/or any carbon metabolite in the productive and/or catabolicpathway, such as by mutation in or deletion of part or all of thepolynucleotide encoding an enzymatic activity that phosphorylates theinitial carbon source and/or any carbon metabolite. The uncoupling ofthe productive pathway and catabolic pathway encompasses regulation ofat least one enzymatic activity that phosphorylates the initial carbonsource and/or any carbon metabolite in the productive and/or catabolicpathway.

One advantage of the invention is that in host cells comprisinguncoupled productive and catabolic pathways, the pathways are able tofunction simultaneously without one pathway creating a disadvantage forthe other. In some embodiments disclosed herein, a host cell having adeletion of glucokinase and gluconokinase is cultured in the presence ofD-glucose. The D-glucose passes through the productive pathway withoutbeing diverted into the catabolic pathway, thereby increasing the amountof carbon substrate available for conversion to the desired productivepathway generated product. Fructose, or other non-glucose carbon source,can be fed to the host cell and is used to satisfy the host cell'smetabolic needs, freeing the D-glucose for use by the product pathwayyielding the desired product. In this embodiment, the productive andcatabolic pathways function simultaneously and non-competitively in thehost cell.

Another advantage of the invention is that in host cells comprisinguncoupled productive and catabolic pathways, either pathway can be usedto provide for the metabolic needs of the host cell, freeing the otherpathway to be used to produce products through that particular pathway.In some embodiments disclosed herein, a host cell having a deletion ofthe coupling enzymes enables the products of the productive pathway tosatisfy the metabolic needs of the host cell, freeing the pathwayusually associated with the generation of energy through the catabolicpathway to generate products. Thus fructose, or other non-glucose carbonsource, can be fed to the host cell and is used to produce derivativesor desired products, while the host cell's metabolic needs are satisfiedby conversion of productive pathway products to metabolic needs in thehost cell.

In other embodiments, the ability of the host cell to use D-glucose, ora metabolite of D-glucose, such as D-gluconate, in the catabolicpathway, that is the ability of the host cell to phosphorylate D-glucoseor D-gluconate at their respective 6th carbons, is regulated. Regulatingthe expression of the enzymatic activity allows a process whereinD-glucose or other carbon source is available to the catabolic pathwayduring the initial phase of culturing, where it is desirable to buildcell biomass, and not available, that is not phosphorylated, at laterstages of culturing where it may be desirable to maximize ATP productionfor use by the cell or where it may be desirable to feed a differentcarbon source to the cell for production of desired product.

In these embodiments, a particular advantage provided by the inventionis the ability to make use of continuous fermentation processes for theproduction of products.

Another advantage provided by the invention is the uncoupling of theextracellular oxidation of a substrate from the metabolic pathways thatuse those oxidation products.

Another advantage provided by the invention is the increased efficiencyin the production of products by the modified host cells as compared towild-type host cells as measured directly by the increased conversion ofsubstrate to end-product or indirectly as measured by O2 consumption orCO₂ production.

A further advantage provided by the invention is the ability of the hostcell to utilize two different carbon sources simultaneously for theproduction of products.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, and biochemistry,which are within the skill of the art. Such techniques are explainedfully in the literature, such as, Molecular Cloning: A LaboratoryManual, second edition (Sambrook et al., 1989); Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., 1987 and annual updates);Oligonucleotide Synthesis (M. J. Gait, ed., 1984); and PCR: ThePolymerase Chain Reaction, (Mullis et al., eds., 1994). Manual ofIndustrial Microbiology and Biotechnology, Second Edition (A. L. Demain,et al., eds. 1999)

Definitions

As used herein, the term “uncoupled” when referring to productive andcatabolic pathways of a host cell means that the productive pathway ofthe host cell, including the substrates and products produced therein,have a reduced diversion of substrates to the catabolic pathway of thehost cell. By reduced diversion, is meant that the yield of the wildtype is less than the yield of the modified host cell.

As used herein, “productive pathway of a host cell” means that the hostcell comprises at least one enzyme that coverts a carbon source, suchas, D-glucose and/or its metabolites to a desired product orintermediate. The productive pathway of the host cell includes but isnot limited to the oxidative pathway of the host cell.

As used herein, “oxidative pathway of a host cell” means that the hostcell comprises at least one oxidative enzyme that oxidizes a carbonsource, such as, D-glucose and/or its metabolites. A “membrane” or“membrane bound” glucose productive pathway in a host cells refers to ahost cell that oxidizes a carbon source such as, D-glucose and/or itsmetabolites, via at least one membrane bound productive enzymaticactivity. In some embodiments, an oxidative pathway in a host cellcomprises one enzymatic activity. In other embodiments, an oxidativepathway in a host cell comprises two or more enzymatic activities.

As used herein, “catabolic pathway of a host cell” means that the hostcell comprises at least one enzymatic activity that generates ATP orNADPH, for example, by phosphorylating a carbon source, such asD-glucose and/or its metabolites. An “intracellular” catabolic pathwayin a host cell means that the host cell comprises at least one suchenzymatic activity in the host cell cytosol. In some embodiments, acatabolic pathway in a host cell comprises one enzymatic activity. Inother embodiments, a catabolic pathway in a host cell comprises two ormore enzymatic activities.

As used herein, the phrase “enzymatic activity which phosphorylatesD-glucose at its 6th carbon” refers to an enzymatic activity that adds aphosphate to the 6th carbon of D-glucose and includes the enzymaticactivities glucokinase (EC-2.7.1.2); and phosphotransferase system (PTS)(E.C.-2.7.1.69). As used herein, the phrase “enzymatic activity whichphosphorylates D-gluconate at its 6th carbon” refers to an enzymaticactivity that phosphorylates D-gluconate at its 6th carbon and includesthe enzymatic activity gluconokinase (E.C.-2.7.1.12).

As used herein, “modifying” the levels of an enzymatic activity producedby a host cell or “modified levels” of an enzymatic activity of a hostcell refers to controlling the levels of enzymatic activity producedduring culturing, such that the levels are increased or decreased asdesired. The desired change in the levels of enzymatic activity may begenetically engineered to take place in one or both enzymatic activitieseither simultaneously or sequentially, in any order. In order to controlthe levels of enzymatic activity, the host cell is geneticallyengineering such that nucleic acid encoding the enzymatic activity istranscriptionally or translationally controlled.

As used herein, the term “modified” when referring to nucleic acid orpolynucleotide means that the nucleic acid has been altered in some wayas compared to wild type nucleic acid, such as by mutation in; deletionof part or all of the nucleic acid; or by being operably linked to atranscriptional control region. As used herein the term “mutation” whenreferring to a nucleic acid refers to any alteration in a nucleic acidsuch that the product of that nucleic acid is partially or totallyinactivated or eliminated. Examples of mutations include but are notlimited to point mutations, frame shift mutations and deletions of partor all of a gene encoding an enzymatic activity, such as an enzymaticactivity that transports the substrate across the cell membrane, e.g.,phosphorylates D-glucose at its 6th carbon or an enzymatic activity thatphosphorylates D-gluconate at its 6th carbon.

An “altered bacterial strain” according to the invention is agenetically engineered bacterial microorganism having an enhanced levelof production over the level of production of the same end-product in acorresponding unaltered bacterial host strain grown under essentiallythe same growth conditions. An “unaltered bacterial strain” or host is abacterial microorganism wherein the coding sequence of the divertingenzymatic pathway is not inactivated and remains enzymatically active.The enhanced level of production results from the inactivation of one ormore chromosomal genes. In a first embodiment the enhanced level ofexpression results from the deletion of one or more chromosomal genes.In a second embodiment the enhanced level of expression results from theinsertional inactivation of one or more chromosomal genes. Preferablythe inactivated genes are selected from those encoding the enzymes whoseinactivity is desired as described elsewhere in this application. Forexample, in one embodiment one or more chromosomal genes is selectedfrom the group consisting of glk, and gntk.

In certain embodiments, the altered bacterial strain may embody twoinactivated genes, three inactivated genes, four inactivated genes, fiveinactivated genes, six inactivated genes or more. The inactivated genesmay be contiguous to one another or may be located in separate regionsof the chromosome. An inactivated chromosomal gene may have a necessaryfunction under certain conditions, but the gene is not necessary formicroorganism strain viability under laboratory conditions. Preferredlaboratory conditions include but are not limited to conditions such asgrowth in a fermentator, in a shake plate, in plate media or the like.

As used herein, the term “inactivation” or “inactivating” when referringto an enzymatic activity means that the activity has been eliminated byany means including a mutation in or deletion of part or all of thenucleic acid encoding the enzymatic activity. The term “inactivation” or“inactivating” includes any method that prevents the functionalexpression of one or more of the desired chromosomal genes, wherein thegene or gene product is unable to exert its known function. The desiredchromosomal genes will depend upon the enzymatic activity that isintended to be inactivated. For example the inactivation of glucokinaseand/or gluconokinase activity can be effected by inactivating the glkand/or gntk chromosomal genes coding regions. Inactivation may includesuch methods as deletions, mutations, interruptions or insertions in thenucleic acid gene sequence. In one embodiment, the expression product ofan inactivated gene may be a truncated protein as long as the truncatedprotein does not show the biological activity of the unaltered codingregion. In an altered bacterial strain according to the invention, theinactivation of the one or more genes will preferably be a stable andnon-reverting inactivation.

In a preferred embodiment, preferably a gene is deleted by homologousrecombination. For example, as shown in FIG. 9, when gik is the gene tobe deleted, a chloramphenicol resistance gene is cloned into a uniquerestriction site found in the glucokinase gene. The Cm^(R) gene isinserted into the structural coding region of the gene at the Pst Isite. Modification is then transferred to the chromosome of a P. citreaglkA- by homologous recombination using a non-repliation R6K vector. TheCm^(R) gene is subsequently removed from the glk coding region leavingan interrupting spacer in the coding region, inactivating the codingregion. In another embodiment, the Cm^(R) gene is inserted into thecoding region in exchange for portions of the coding region. Subsequentremoval of the Cm^(R) gene without concomitant return of the exchangedout portion of the coding region results in an effective deletion of aportion of the coding region, inactivating such region.

A deletion of a gene as used herein may include deletion of the entirecoding sequence, deletion of part of the coding sequence, or deletion ofthe coding sequence including flanking regions. The deletion may bepartial as long as the sequences left in the chromosome are too shortfor biological activity of the gene. The flanking regions of the codingsequence may include from about 1 bp to about 500 bp at the 5′ and 3′ends. The flanking region may be larger than 500 bp but will preferablynot include other genes in the region which may be inactivated ordeleted according to the invention. The end result is that the deletedgene is effectively non-functional.

In another preferred embodiment, inactivation is by insertion. Forexample when glk is the gene to be inactivated, a DNA construct willcomprise an incoming sequence having the glk gene interrupted by aselective marker. The selective marker will be flanked on each side bysections of the glk coding sequence. The DNA construct aligns withessentially identical sequences of the glk gene in the host chromosomeand in a double crossover event the glk gene is inactivated by theinsertion of the selective marker.

In another embodiment, inactivation is by insertion in a singlecrossover event with a plasmid as the vector. For example, a glkchromosomal gene is aligned with a plasmid comprising the gene or partof the gene coding sequence and a selective marker. The selective markermay be located within the gene coding sequence or on a part of theplasmid separate from the gene. The vector is integrated into theBacillus chromosome, and the gene is inactivated by the insertion of thevector in the coding sequence.

Inactivation may also occur by a mutation of the gene. Methods ofmutating genes are well known in the art and include but are not limitedto chemical mutagenesis, site-directed mutation, generation of randommutations, and gapped-duplex approaches. (U.S. Pat. No. 4,760,025;Moring et al., Biotech. 2:646 (1984); and Kramer et al., Nucleic AcidsRes. 12:9441 (1984)).

Inactivation may also occur by applying the above described inactivationmethods to the respective promoter regions of the desired genomicregion.

“Under transcriptional control” or “transcriptionally controlled” areterms well understood in the art that indicate that transcription of apolynucleotide sequence, usually a DNA sequence, depends on its beingoperably (operatively) linked to an element which contributes to theinitiation of, or promotes, transcription. “Operably linked” refers to ajuxtaposition wherein the elements are in an arrangement allowing themto function.

As used herein, the term “regulatable promoter” refers to a promoterelement which activity or function can be modulated. This modulation canbe accomplished in many different ways, most commonly by the interactionof protein(s) that interfere or increase the ability of the RNApolymerase enzyme to initiate transcription.

“Under translational control” well understood in the art that indicatesa regulatory process that occurs after the messenger RNA has beenformed.

As used herein, the term “batch” describes a batch cell culture to whichsubstrate, in either solid or concentrated liquid form, is addedinitially at the start of the run. A batch culture is initiated byinoculating cells to the medium, but, in contrast to a fed-batchculture, there is no subsequent inflow of nutrients, such as by way of aconcentrated nutrient feed. In contrast to a continuous culture, in abatch cell culture, there is no systematic addition or systematicremoval of culture fluid or cells from a culture. There is no ability tosubsequently add various analytes to the culture medium, since theconcentrations of nutrients and metabolites in culture medium aredependent upon the initial concentrations within the batch and thesubsequent alteration of the composition of the nutrient feed due to theact of fermentation.

As used herein, the term “fed-batch” describes a batch cell culture towhich substrate, in either solid or concentrated liquid form, is addedeither periodically or continuously during the run. Just as in a batchculture, a fed-batch culture is initiated by inoculating cells to themedium, but, in contrast to a batch culture, there is a subsequentinflow of nutrients, such as by way of a concentrated nutrient feed. Incontrast to a continuous culture there is no systematic removal ofculture fluid or cells from a fed-batch culture is advantageous inapplications that involve monitoring and manipulating the levels ofvarious analytes in the culture medium, since the concentrations ofnutrients and metabolites in culture medium can be readily controlled oraffected by altering the composition of the nutrient feed. The nutrientfeed delivered to a fed-batch culture is typically a concentratednutrient solution containing an energy source, e.g., carbohydrates;optionally, the concentrated nutrient solution delivered to a fed-batchculture can contain amino acids, lipid precursors and/or salts. In afed-batch culture, this nutrient feed is typically rather concentratedto minimize the increase in culture volume while supplying sufficientnutrients for continued cell growth.

The term “continuous cell culture” or, simply, “continuous culture” isused herein to describe a culture characterized by both a continuousinflow of a liquid nutrient feed and a continuous liquid outflow. Thenutrient feed may, but need not, be a concentrated nutrient feed.Continuously supplying a nutrient solution at about the same rate thatcells are washed out of the reactor by spent medium allows maintenanceof a culture in a condition of stable multiplication and growth. In atype of bioreactor known as a chemostat, the cell culture iscontinuously fed fresh nutrient medium, and spent medium, cells andexcreted cell product are continuously drawn off. Alternatively, acontinuous culture may constitute a “perfusion culture,” in which casethe liquid outflow contains culture medium that is substantially free ofcells, or substantially lower cell concentration than that in thebioreactor. In a perfusion culture, cells can be retained by, forexample, filtration, centrifugation, or sedimentation.

“Culturing” as used herein refers to fermentive bioconversion of acarbon substrate to the desired end-product within a reactor vessel.Bioconversion as used herein refers to the use of contacting amicroorganism with the carbon substrate to convert the carbon substrateto the desired end-product.

As used herein, “Oxygen Uptake Rate or “OUR” refers to the determinationof the specific consumption of oxygen within the reactor vessel. Oxygenconsumption can be determined using various on-line measurements. In oneexample, the OUR (mmol/(liter*hour)) is determined by the followingformula:((Airflow (standing liters per minute)/Fermentation weight (weight ofthe fermentation broth in kilograms))×supply O₂×broth density×(aconstant to correct for airflow calibration at 21.1 C instead ofstandard 20.0 C)) minus ([airflow/fementation weight]×[offgas O₂/offgasN₂]×supply N₂×broth density×constant).

As used herein, “carbon evolution rate or “CER” refers to thedetermination of how much CO₂ is produced within the reactor vesselduring fermentation. Usually, since no CO₂ is initially or subsequentlyprovided to the reaction vessel, any CO₂ is assumed to be produced bythe fermentation process occurring within the reaction vessel. “Off-gasCO₂” refers to the amount of CO₂ measured within the reactor vessel,usually by mass spectroscopic methods known in the art.

As used herein, “yield” refers to the amount of product divided by theamount of substrate. The yield can be expressed as a weight % (productgm/substrate gm) or as moles of product/moles of substrate. For example,the amount of the substrate, e.g., glucose can be determined by the feedrate and the concentration of the added glucose. The amount of productspresent can be determined by various spectrophotometric or analyticmethodologies. One such methodology is high performance liquidchromatography (HPLC). An increased yield refers to an increased yieldas compared to the yield of a conversion using the wild-type organism,for example an increase of 10%, 20%, or 30% over the yield of thewild-type.

The phrase “oxidative enzyme” as used herein refers to an enzyme orenzyme system which can catalyze conversion of a substrate of a givenoxidation state to a product of a higher oxidation state than substrate.The phrase “reducing enzyme” refers to an enzyme or enzyme system whichcan catalyze conversion of a substrate of a given oxidation state to aproduct of a lower oxidation state than substrate. In one illustrativeexample disclosed herein, productive enzymes associated with thebiocatalysis of D-glucose or its metabolites in a Pantoea cell which hasbeen engineered to produce ASA intermediates, include among othersD-glucose dehydrogenase, D-gluconate dehydrogenase and2-keto-D-gluconate dehydrogenase. In another illustrative embodimentdisclosed herein, reducing enzymes associated with the biocatalysis ofD-glucose or its metabolites in a Pantoea cell which has been engineeredto produce ASA intermediates, as described herein, include among others2,5-diketo-D-gluconate reductase, 2-keto reductase and 5-keto reductase.Such enzymes include those produced naturally by the host strain orthose introduced via recombinant means.

As used herein, the term carbon source encompasses suitable carbonsources ordinarily used by microorganisms, such as 6 carbon sugars,including but not limited to glucose, gulose, sorbose, fructose, idose,galactose and mannose all in either D or L form, or a combination of 6carbon sugars, such as glucose and fructose, and/or 6 carbon sugar acidsincluding but not limited to 2-keto-L-gulonic acid, idonic acid,gluconic acid, 6-phosphogluconate, 2-keto-D-gluconic acid,5-keto-D-gluconic acid, 2-ketogluconatephosphate, 2,5-diketo-L-gulonicacid, 2,3-L-diketogulonic acid, dehydroascorbic acid, erythroascorbicacid, erythorbic acid and D-mannonic acid or the enzymatic derivativesof such.

The following definitions apply as used herein to D-glucose or glucose(G); D-gluconate or gluconate (GA); 2-keto-D-gluconate (2 KDG);2,5-diketo-D-gluconate (2,5DKG or DKG); 2-keto-L-gulonic acid (2KLG, orKLG); L-idonic acid (IA); erythorbic acid (EA); ascorbic acid (ASA);glucose dehydrogenase (GDH); gluconic acid dehydrogenase (GADH);2,5-diketo-D-gluconate reductase (DKGR); 2-keto-D-gluconate reductase(KDGDH); D-ribose (R); 2-keto reductase (2KR or KR); and 5-ketoreductase (5KR or KR).

“Carbon metabolite” as used herein refers to a compound that is utilizedin the catabolic pathway to generate ATP, NADPH and/or is phosphorylatedfor transport into the cell.

“Allowing the production of an ascorbic acid intermediate from thecarbon source, wherein the production of said ascorbic acid intermediateis enhanced compared to the production of the ascorbic acid intermediatein the unaltered bacterial host strain” means contacting the substrate,e.g. carbon source, with the altered bacterial strain to produce thedesired end-product. The inventors discovered that by altering certainenzymatic activities by inactivating genomic expression, themicroorganism demonstrated enhanced end-product production.

“Desired end-product” as used herein refers to the desired compound towhich the carbon substrate is bioconverted into. The desired end-productmay be the actual compound sought or an intermediate along anotherpathway. Exemplary desired end-products are listed on the right side ofFIG. 3.

As used herein, the term “bacteria” refers to any group of microscopicorganisms that are prokaryotic, i.e., that lack a membrane-bound nucleusand organelles. All bacteria are surrounded by a lipid membrane thatregulates the flow of materials in and out of the cell. A rigid cellwall completely surrounds the bacterium and lies outside the membrane.There are many different types of bacteria, some of which include, andare not limited to those strains within the families ofEnterobacteriaceae, Bacillus, Streptomyces, Pseudomonas, and Erwinia.

As used herein, the family “Enterobacteriaceae” refers to bacterialstrains having the general characteristics of being Gram negative andbeing facultatively anaerobic. For the production of ASA intermediates,preferred Enterobacteriaceae strains are those that are able to produce2,5-diketo-D-gluconic acid from D-glucose or carbon sources which can beconverted to D-glucose by the strain. Included in the family ofEnterobacteriaceae which are able to produce 2,5-diketo-D-gluconic acidfrom D-glucose solutions are the genus Erwinia, Enterobacter,Gluconobacter and Pantoea, for example. Intermediates in the microbialpathway from carbon source to ASA, include but are not limited to GA,KDG, DKG, DKG, KLG and IA. In the present invention, a preferredEnterobacteriaceae fermentation strain for the production of ASAintermediates is a Pantoea species and in particular, Pantoea citrea.

As used herein the family “Bacillus” refers to rod-shaped bacterialstrains having the general characteristics of being gram positive,capable of producing spores under certain environmental conditions.Other Enterobacteriaceae strains that produce ASA intermediates include,but are not limited to, E. coli and Gluconobacter.

As used herein, the term “recombinant” refers to a host cell that has amodification of its genome, eg as by the additional of nucleic acid notnaturally occurring in the organism or by a modification of nucleic acidnaturally occurring in the host cell and includes host cells havingadditional copies of endogenous nucleic acid introduced via recombinantmeans. The term “heterologous” as used herein refers to nucleic acid oramino acid sequences not naturally occurring in the host cell. As usedherein, the term “endogenous” refers to a nucleic acid naturallyoccurring in the host.

The terms “isolated” or “purified” as used herein refer to an enzyme, ornucleic acid or protein or peptide or co-factor that is removed from atleast one component with which it is naturally associated. In thepresent invention, an isolated nucleic acid can include a vectorcomprising the nucleic acid.

It is well understood in the art that the acidic derivatives ofsaccharides, may exist in a variety of ionization states depending upontheir surrounding media, if in solution, or out of solution from whichthey are prepared if in solid form. The use of a term, such as, forexample, idonic acid, to designate such molecules is intended to includeall ionization states of the organic molecule referred to. Thus, forexample, “idonic acid”, its cyclized form “idonolactone”, and “idonate”refer to the same organic moiety, and are not intended to specifyparticular ionization states or chemical forms.

As used herein, the term “vector” refers to a polynucleotide constructdesigned for transduction/transfection of one or more cell typesincluding for example, “cloning vectors” which are designed forisolation, propagation and replication of inserted nucleotides or“expression vectors” which are designed for expression of a nucleotidesequence in a host cell, such as a Pantoea citrea or E. coli host cell.

The terms “polynucleotide” and “nucleic acid”, used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. These terms include a single-,double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid,or a polymer comprising purine and pyrimidine bases, or other natural,chemically, biochemically modified, non-natural or derivatizednucleotide bases. The backbone of the polynucleotide can comprise sugarsand phosphate groups (as may typically be found in RNA or DNA), ormodified or substituted sugar or phosphate groups. Alternatively, thebackbone of the polynucleotide can comprise a polymer of syntheticsubunits such as phosphoramidates and thus can be a oligodeoxynucleosidephosphoramidate (P-NH2) or a mixed phosphoramidate-phosphodiesteroligomer. Peyrottes et al. (1996) Nucleic Acids Res. 24: 1841-8;Chaturvedi et al. (1996) Nucleic Acids Res. 24: 2318-23; Schultz et al.(1996) Nucleic Acids Res. 24: 2966-73. A phosphorothioate linkage can beused in place of a phosphodiester linkage. Braun et al. (1988) J.Immunol. 141: 2084-9; Latimer et al. (1995) Molec. Immunol. 32:1057-1064. In addition, a double-stranded polynucleotide can be obtainedfrom the single stranded polynucleotide product of chemical synthesiseither by synthesizing the complementary strand and annealing thestrands under appropriate conditions, or by synthesizing thecomplementary strand de novo using a DNA polymerase with an appropriateprimer. Reference to a polynucleotide sequence (such as referring to aSEQ ID NO) also includes the complement sequence.

The following are non-limiting examples of polynucleotides: a gene orgene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs,uracyl, other sugars and linking groups such as fluororibose andthioate, and nucleotide branches. The sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component. Other types of modifications included in thisdefinition are caps, substitution of one or more of the naturallyoccurring nucleotides with an analog, and introduction of means forattaching the polynucleotide to proteins, metal ions, labelingcomponents, other polynucleotides, or a solid support. Preferably, thepolynucleotide is DNA. As used herein, “DNA” includes not only bases A,T, C, and G, but also includes any of their analogs or modified forms ofthese bases, such as methylated nucleotides, internucleotidemodifications such as uncharged linkages and thioates, use of sugaranalogs, and modified and/or alternative backbone structures, such aspolyamides.

A polynucleotide or polynucleotide region has a certain percentage (forexample, 80%, 85%, 90%, 95%, 97% or 99%) of “sequence identity” toanother sequence means that, when aligned, that percentage of bases arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., 1987) Supplement 30,section 7.7.18. A preferred alignment program is ALIGN Plus (Scientificand Educational Software, Pennsylvania), preferably using defaultparameters, which are as follows: mismatch=2; open gap=0; extend gap=2.

A polynucleotide sequence that is “depicted in” a SEQ ID NO means thatthe sequence is present as an identical contiguous sequence in the SEQID NO. The term encompasses portions, or regions of the SEQ ID NO aswell as the entire sequence contained within the SEQ ID NO.

“Expression” includes transcription and/or translation.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense; that is, equivalent to the term “including” and itscorresponding cognates.

“A,” “an” and “the” include plural references unless the context clearlydictates otherwise.

Productive and Catabolic Pathways of Host Cells

FIGS. 2 and 3 describe some of the products of metabolism that can beobtained from some of the metabolic routes. The majority of the productson the left side of FIG. 3 (Glucose-6-phosphate, glucose-1-phosphate;fructose-6-phosphate, mannose-6-phosphate, dihydroacetone-phosphate;dihydroacetone; glycerol; 1,2-propanediol; 1,3 propanediol; lactic acid;succinic acid; oxalic acid; citric acid; fumaric acid; malic aicd; aminoacids; glycogen; trehalose; and UDP-glucose) can be obtained from thecatabolic or TCA cycle. On the contrary, the compounds on the right,desired end-products for the purposes of this invention (gluconic acid,2-keto-D-gluconic acid, 2,5-di-keto-gluconate; erythorbic acid;5-keto-D-gluconate; tartaric acid; D-ribose; riboflavin;deoxyribonucleotides; aromatic amino aicds, aromatic compounds [e.g.P-hydroxybenzoic acid; quinines; catechols; indoles; indigo; gallicacid; pyrogallol; melanin, adipic acid, p-aminobenzoic acid]; pyridoxineand aspartame) derive most of its carbon from the pentose pathway and/orfrom the oxidation of glucose into keto acid. In many cases, theseproducts are not natural products of the metabolism of a particularcell, but they can be produced by adding or removing certain enzymaticfunctions.

Generally, those products on the left side of FIG. 3 are used tomaintain the catabolic needs of the host cell. By uncoupling theinteraction between those compounds on the left with those on the right,the metabolic requirements of the host cell are satisfied by theproducts generated on one side, enabling more carbon substrate to beconverted into the desired productive product. In one embodiment, theuncoupling of the productive pathways from the catabolic pathwaysincrease the yield of compounds produced on the right side. In anotherembodiment, it is contemplated using the products generated by theproductive pathways to maintain the metabolic requirements of the hostcell would enable those reactions in the catabolic pathways to beutilized to increase the yield of products derived from those productswithin the catabolic pathway, e.g. 1,3-propanediol, DHAP, lactic acid.

The invention also includes functionally-preserved variants of themodified nucleic acid sequences disclosed herein, which include nucleicacid substitutions, additions, and/or deletions. In one embodiment, thevariants include modified sequences encoding glucokinase andgluconokinase, which inactivates the enzymatic pathway convertingglucose to glucose-6-phosphate and gluconate to gluconate-6-phosphate,uncoupling the productive pathways from the catabolic pathways, reducingthe amount of carbon substrate diverted to the catabolic pathway andincreasing the amount of carbon substrate available for conversion intothe desired product, e.g. 2-KLG. Genetic modifications are used toeliminate the communication between the catabolic functions and theenzymatic reactions that are required to synthesize a desired product.While various modifications are described in this application (see FIGS.17-24), the inventors contemplate that other enzymatic steps could bemodified to achieve the same uncoupling oxidative, catabolic pathwayuncoupling.

Esters of phosphoric acid are encountered with trioses, tetroses,pentoses, hexoses and heptoses. The phosphorylation of all sugars is theinitial step in their metabolism. Thus glucose can be phosphorylated toglucose 6-phospahte. All cells that can metabolize glucose contain someform of a hexokinase which catalyze the reaction

FIG. 9 depicts D-glucose and illustrates the “6th carbon”. Exemplaryhexokinases include hexokinase (Frohlich, et al., 1985, Gene 36:105-111)and glucokinase (Fukuda, et al., 1983, J. Bacteriol. 156:922-925). TheDNA sequence of the glucokinase structural gene from P. citrea is shownin FIG,. 4. The recogition site for the restriction enzymes Ncol(CCATGG) and SnaBI (TACGTA) are highlighted. FIG. 5 depicts the proteinsequence of the glucokinase gene from P. citrea. Most hexokinases aresomewhat nonspecific, showing some ability to catalyze formation of6-phosphate esters of mannose, fructose, and galactose. In addition,other hexose derivatives may also be phosphorylated by a hexokinase.Gluconate (FIG. 3), for example, may also be phosphorylated by a kinase,specifically gluconokinase (citation). The sequence for thegluconokinase structural gene from P. citrea is depicted in FIG. 6. Therecognition site for the restriction enzyme Pst I (CTGCAG) ishighlighted. The protein sequence for the gluconokinase gene from P.citrea is depicted in FIG. 7 (SEQ ID NO 4). The some of the genes forglucokinase And gluconokinase (glk, gntk, etc.) are shown in FIG. 8.

FIG. 17 shows the interrelationships between the catabolic pathways andthe productive (oxidative) pathway. Glucose can enter the catabolicpathways through the glycolytic pathway by the phosphorylation ofglucose to glucose-6-phosphate by glucokinase (Glk); and through thepentose pathway by the phosphorylation of gluconate togluconate-6-phosphate by glucono kinase (Gntk). Inactivation ormodifying the levels of glucokinase and gluconokinase by modifying thenucleic acid or polypeptide encoding the same (glk or gntk), results inthe increased yield of the desired product, e.g. an ascorbic acidintermediate. As used herein, ascorbic acid intermediate includes thosesugar acids produced within the oxidative pathway from glucose to 2KLG,including, but not limited to gluconate, 2-KGD, 2,5-DKG, 2-KLG, and5-DKG.

In another embodiment, the catabolic pathway is uncoupled from theproductive pathway to increase the production of ribose. As shown inFIG. 18, glucose can enter the catabolic pathway through the glycolyticpathway, for example through glucose-6-phosphate, fructose-6-phosphate,and/or glyceraldehydes-3-phosphate. Inactivation or modifying the levelsof glucokinase, gluconokinase, ribulose-5-phosphate epimerase,transketolase and transaldolase, by modifying the nucleic acid orpolypeptide encoding the same, results in the increased yield of thedesired product, e.g. ribose.

In another embodiment, the catabolic pathway is uncoupled from theproductive pathway to increase the production of riboflavin. As shown inFIG. 19, glucose can enter the catabolic pathway through the glycolyticpathway, for example through glucose-6-phosphate, and the pentosepathway. Inactivation or modifying the levels of glucokinase,ribulose-5-phosphate epimerase and ribose-5-phosphate isomerase, bymodifying the nucleic acid or polypeptide encoding the same, results inthe increased yield of the desired product, e.g. riboflavin.

In another embodiment, the catabolic pathway is uncoupled from theproductive pathway to increase the production of nucleotides. As shownin FIG. 20, glucose can enter the catabolic pathway through theglycolytic pathway, for example through glucose-6-phosphate,fructose-6-phosphate, and/or glyceraldehydes-3-phosphate. Inactivationor modifying the levels of glucokinase, ribulose-5-phosphate epimerase,transaldolase and transketolase, by modifying the nucleic acid orpolypeptide encoding the same, results in the increased yield of thedesired product, e.g. nucleotides.

In another embodiment, the catabolic pathway is uncoupled from theproductive pathway to increase the production of 5-KDG and/or tartrate.As shown in FIG. 21, glucose can enter the catabolic pathway through theglycolytic pathway, for example through glucose-6-phosphate, the pentosepathway through gluconate-6-phosphate, and other ascorbic acidby-products, such as idonate and 2-KLG. Inactivation or modifying thelevels of glucokinase, gluconokinase, 2,5-DKG reductase, and Idonatedehydrogenase, by modifying the nucleic acid or polypeptide encoding thesame, results in the increased yield of the desired product, e.g. 5-DKGand/or tartrate.

In another embodiment, the catabolic pathway is uncoupled from theproductive pathway to increase the production of gluconate. As shown inFIG. 22, glucose can enter the catabolic pathway through the glycolyticpathway, for example through glucose-6-phosphate and the pentosepathway, through gluconate-6-phosphate. Inactivation or modifying thelevels of glucokinase, gluconokinase, and glyceraldhehyde hydrogenase,by modifying the nucleic acid or polypeptide encoding the same, resultsin the increased yield of the desired product, e.g. gluconate.

In another embodiment, the catabolic pathway is uncoupled from theproductive pathway to increase the production of erythorbic acid. Asshown in FIG. 23, glucose can enter the catabolic pathway through theglycolytic pathway, for example through glucose-6-phosphate; the pentosepathway, through gluconate-6-phosphate; and by an enzymatic transportsystem transporting 2-KDG and 2,5-KDG into the cytoplasm. Inactivationor modifying the levels of glucokinase, gluconokinase, glyceraldhehydehydrogenase and the transport system of 2-KDG into the cytoplasm, bymodifying the nucleic acid or polypeptide encoding the same, results inthe increased yield of the desired product, e.g. erythoric acid.

In another embodiment, the catabolic pathway is uncoupled from theproductive pathway to increase the production of 2,5-DKG. As shown inFIG. 24, glucose can enter the catabolic pathway through the glycolyticpathway, for example through glucose-6-phosphate; the pentose pathway,through gluconate-6-phosphate; and by an enzymatic transport systemtransporting 2-KDG and 2,5-KDG into the cytoplasm. Inactivation ormodifying the levels of glucokinase, gluconokinase, and 2-KDGhydrogenase; and the enzymatic transport system for 2-KDG, by modifyingthe nucleic acid or polypeptide encoding the same, results in theincreased yield of the desired product, e.g. 2,5-DKG. Wherein theinventors have provided in some instances amino acid sequences andnucleotide sequences for genomic coding regions and/or protein (enzymes)in question, those skilled in the art will recognize that the genomicloci not specifically provided herein are readily ascertainable byconstruction of probes or hybridizing sequences incorporating alreadyknown sequences and a homology alignment of (for example BLAST), in oneembodiment, at least 30% or at least 50%, another embodiment, of theknown coding region sequence. In another embodiment, a homologyalignment of at least 60%, 70&, 75%, 80%, 90%, 95%, 97% or even 98% ofthe known sequence will identify the coding region to which theinactivation techniques described elsewhere are applied to effect thedesired. Another methodology to determine the coding regions for theparticular enzyme known to those of skill in the art is to obtainseveral known sequences, align the sequences to determine the conservedregion, then design degenerate oligoprimers followed by PCRamplification of the connecting regions between the framing residues toascertain the desired genomic region.

The availability of recombinant techniques to effect expression ofenzymes in foreign hosts permits the achievement of the aspect of theinvention which envisions production of a desired end-product, e.g.,riboflavin, tartrate, 5-KDG, ribose, nucleotides, gluconate, erythorbicacid, 2,5-DKG, other ascorbic acid intermediates or other desiredproducts with a reduced amount of carbon substrate diverted to catabolicpathways from a readily available carbon substrate. This method hasconsiderable advantage over presently used methods in characterized by areduction in the amount of substrate converted to the catabolic pathwayand thus unavailable for conversion to the desired oxidativeend-product, e.g., an ascorbic acid intermediate. This results inincreased fermentative efficiency and increased yield over fermentationswith wild type organisms. Certain wild type organisms may produceascorbic acid intermediates, e.g., 2-KLG, however the level produced maynot be sufficient to be economically practical. It has been observedthat wild type Pantoea citrea has its own cytoplasmic glucokinase andgluconokinase enabling the organism to convert glucose to phosphorylatedderivatives for use in its central metabolic pathways and the productionof which, necessarily consume energy, ATP and causes that more carbongoes to non-2-KLG producing pathways. Under the same controlledconditions and using the method of this invention, described below, intwo interruption plasmid described elsewhere in this application theglucokinase and gluconokinase genes can be deleted from the P. citreagenome, enabling the modified P. citrea to produce increased DKG fromglucose at a a level increased over the wild-type, e.g.level of 63%yield to about 97-98% yield. [see Example 6].

Other variants include, but are not limited to, inactivations of the gapgene to increase production of dihydroacetone-phosphate, DHAP;erythorbic acid; and tartic acid.

The variants of the sequences disclosed herein may be 80%, 85%, 90%,95%, 98%, 99% or more identical, as measured by, for example, ALIGN Plus(Scientific and Educational Software, Pennsylvania), preferably usingdefault parameters, which are as follows: mismatch=2; open gap=0; extendgap=2 to any of the enzymatic sequences disclosed herein. Variants ofglucokinase and gluconokinase sequences may also hybridize at highstringency, that is at 68° C. and 0.1×SSC, to the glucokinase andgluconokinase sequences disclosed herein.

In terms of hybridization conditions, the higher the sequence identityrequired, the more stringent are the hybridization conditions if suchsequences are determined by their ability to hybridize to a sequence ofSEQ ID NO:1 or SEQ ID NO:3. Accordingly, the invention also includespolynucleotides that are able to hybridize to a sequence comprising atleast about 15 contiguous nucleotides (or more, such as about 25, 35,50, 75 or 100 contiguous nucleotides) of SEQ ID NO:1 or SEQ ID NO:3. Thehybridization conditions would be stringent, i.e., 80° C. (or highertemperature) and 6M SSC (or less concentrated SSC). Another set ofstringent hybridization conditions is 68° C. and 0.1×SSC. For discussionregarding hybridization reactions, see below.

Hybridization reactions can be performed under conditions of different“stringency”. Conditions that increase stringency of a hybridizationreaction of widely known and published in the art. See, for example,Sambrook et al. (1989) at page 7.52. Examples of relevant conditionsinclude (in order of increasing stringency): incubation temperatures of25° C., 37° C., 50° C. and 68° C.; buffer concentrations of 10×SSC,6×SSC, 1×SSC, 0.1×SSC (where SSC is 0.15 M NaCl and 15 mM citratebuffer) and their equivalents using other buffer systems; formamideconcentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutesto 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2,or 15 minutes; and wash solutions of 6×SSC, 1×SSC, 0.1×SSC, or deionizedwater. An exemplary set of stringent hybridization conditions is 68° C.and 0.1×SSC.

“T_(m)” is the temperature in degrees Celcius at which 50% of apolynucleotide duplex made of complementary strands hydrogen bonded inanti-parallel direction by Watson-Crick base pairing dissociates intosingle strands under conditions of the experiment. T_(m) may bepredicted according to a standard formula, such as:T _(m)=81.5+16.6 log[X ⁺]+0.41 (%G/C)−0.61 (%F)−600/Lwhere [X⁺] is the cation concentration (usually sodium ion, Na⁺) inmol/L; (% G/C) is the number of G and C residues as a percentage oftotal residues in the duplex; (% F) is the percent formamide in solution(wt/vol); and L is the number of nucleotides in each strand of theduplex.

I. Production of ASA Intermediates

The present invention also provides methods for the production ofascorbic acid intermediates in host cells. The present inventionencompasses methods wherein the levels of an enzymatic activity couplethe catabolic and productive pathways, e.g., those which phosphorylateD-glucose at its 6th carbon and/or which phosphorylates D-gluconate atits 6th carbon are decreased during part or all of the culturing. Thepresent invention encompasses methods wherein the levels of an enzymaticactivity which phosphorylates D-glucose at its 6th carbon and/or thelevels of an enzymatic activity which phosphorylates D-gluconate at its6th carbon are increased during part or all of the culturing. Thepresent invention also encompasses a method wherein the levels of anenzymatic activity which phosphorylates D-glucose as its 6th carbonand/or the levels of an enzymatic activity which phosphorylatesD-gluconate at its 6th carbon are not modified or are increased at thebeginning of the culturing to facilitate growth, that is, to producecell biomass, and decreased during the later phases of culturing tofacilitate desired product accumulation.

The ASA intermediate may be further converted to a desired end productsuch as ASA or erythorbate. For the production of ASA intermediates, anyhost cell which is capable of converting a carbon source to DKG can beused. Preferred strains of the family Enterobacteriaceae are those thatproduce 2,5-diketo-D-gluconic acid from D-glucose solutions, includingPantoea, are described in Kageyama et al. (1992) International Journalof Systematic Bacteriology vol.42, p.203-210. In a preferred embodiment,the host cell is Pantoea citrea having a deletion of part or all of apolynucleotide that encodes an endogenous glucokinase (encoded bynucleic acid as depicted in SEQ ID NO:1) and a deletion of part or allof a polynucleotide that encodes an endogenous gluconokinase (encoded bynucleic acid as depicted in SEQ ID NO:3).

The production of ASA intermediates can proceed in a fermentativeenvironment, that is, in an in vivo environment, or a non-fermentativeenvironment, that is, in an in vitro environment; or combined in vivolinvitro environments. In the methods which are further described infra,the host cell or the in vitro environment further comprise aheterologous DKG reductase which catalyses the conversion of DKG to KLG.

A. In vivo Biocatalytic Environment

The present invention encompasses the use of host cells comprising amodification in a polynucleotide encoding an endogenous enzymaticactivity that phosphorylates D-glucose at its 6th carbon and/or amodification in a polynucleotide encoding an enzymatic activity thatphosphorylates D-gluconate at its 6th carbon in the in vivo productionof ASA intermediates. Biocatalysis begins with culturing the host cellin an environment with a suitable carbon source ordinarily used byEnterobacteriaceae strains, such as a 6 carbon sugar, for example,glucose, or a 6 carbon sugar acid, or combinations of 6 carbon sugarsand/or 6 carbon sugar acids. Other carbon sources include, but are notlimited to galactose, lactose, fructose, or the enzymatic derivatives ofsuch. In addition to an appropriate carbon source, fermentation mediamust contain suitable minerals, salts, cofactors, buffers and othercomponents, known to those of skill in the art for the growth ofcultures and promotion of the enzymatic pathway necessary for productionof desired end-products.

In one illustrative in vivo Pantoea pathway, D-glucose undergoes aseries of membrane productive steps through enzymatic conversions, whichmay include the enzymes D-glucose dehydrogenase, D-gluconatedehydrogenase and 2-keto-D-gluconate dehydrogenase to give intermediateswhich may include, but are not limited to GA, KDG, and DKG, see FIG. 1.These intermediates undergo a series of intracellular reducing stepsthrough enzymatic conversions, which may include the enzymes2,5-diketo-D-gluconate reductase (DKGR), 2-keto reductase (2-KR) and5-keto reductase (5-KR) to give desired end products which include butare not limited to KLG and IA. In a preferred embodiment of the in vivoenvironment for the production of ASA intermediates, 5-KR activity isdeleted in order to prevent the consumption of IA

If KLG is a desired intermediate, nucleic acid encoding DKGR isrecombinantly introduced into the Pantoea fermentation strain. Manyspecies have been found to contain DKGR particularly members of theCoryneform group, including the genera Corynebacterium, Brevibacterium,and Arthrobacter.

In some embodiments of the present invention, 2,5-DKGR fromCorynebacterium sp. strain SHS752001 (Grindley et al., 1988, Applied andEnvironmental Microbiology 54: 1770-1775) is recombinantly introducedinto a Pantoea strain. Production of recombinant 2,5 DKG reductase byErwinia herbicola is disclosed in U.S. Pat. No. 5,008,193 to Anderson etal. Other sources of DKG reductase are provided in Table I.

The fermentation may be performed in a batch process or in a continuousprocess. In a batch process, regardless of what is added, all of thebroth is harvested at the same time. In a continuous system, the brothis regularly removed for downstream processing while fresh substrate isadded. The intermediates produced may be recovered from the fermentationbroth by a variety of methods including ion exchange resins, absorptionor ion retardation resins, activated carbon,concentration-crystallization, passage through a membrane, etc.

B. In vitro Biocatalytic Environment

The invention provides for the biocatalytic production of ASAintermediates, e.g., KDG, DKG and KLG, from a carbon source in an invitro or non-fermentative environment, such as in a bioreactor. Thecells are first cultured for growth and for the non-fermentative processthe carbon source utilized for growth is eliminated, the pH ismaintained at between about pH 4 and about pH 9 and oxygen is present.

Depending upon the desired intermediate being produced, the process mayrequire the presence of enzymatic co-factor. In a preferred embodimentdisclosed herein, the enzymatic co-factor is regenerated. In someembodiments, KDG is the desired ASA intermediate produced, thebioreactor is provided with viable or non-viable Pantoea citrea hostcells comprising a modification in a polynucleotide encoding anendogenous enzymatic activity that phosphorylates D-glucose at its 6thcarbon and/or a modification in a polynucleotide encoding an enzymaticactivity that phosphorylates D-gluconate at its 6th carbon . In thisembodiment, the host cell also has a mutation in a gene encoding2-keto-D-gluconate dehydrogenase activity. In this embodiment, thecarbon source is biocatalytically converted through two productivesteps, to KDG. In this embodiment, there is no need for co-factorregeneration.

When DKG is the desired ASA intermediate, the bioreactor is providedwith viable or non-viable Pantoea citrea host cells comprising amodification in a polynucleotide encoding an endogenous enzymaticactivity that phosphorylates D-glucose at its 6th carbon and/or amodification in a polynucleotide encoding an enzymatic activity thatphosphorylates D-gluconate at its 6th carbon and a carbon source whichis biocatalytically converted through three productive steps, to DKG. Inthis embodiment, there is no need for co-factor regeneration.

When KLG is the desired ASA intermediate, the bioreactor is providedwith viable or non-viable Pantoea citrea host cells comprising amodification in a polynucleotide encoding an endogenous enzymaticactivity that phosphorylates D-glucose at its 6th carbon and/or amodification in a polynucleotide encoding an enzymatic activity thatphosphorylates D-gluconate at its 6th carbon and a carbon source, suchas D-glucose, which is biocatalytically converted through threeproductive steps, and one reducing step to KLG. In this embodiment, thereductase activity may be encoded by nucleic acid contained within thePantoea citrea host cell or provided exogenously. In this embodiment,the first productive enzymatic activity requires an oxidized form of theco-factor and the reducing enzymatic activity requires a reduced form ofco-factor. In a preferred embodiment disclosed herein, the Pantoeacitrea cell is modified to eliminate the naturally occurring GDHactivity and a heterologous GDH activity, such as one obtainable from T.acidophilum, Cryptococcus uniguttalatus or Bacillus species and having aspecificity for NADPH+, is introduced into the Pantoea cell in order toprovide a co-factor recycling system which requires and regenerates oneco-factor. In this embodiment, the host cell further comprises nucleicacid encoding a 2,5-DKG reductase activity or the 2,5-DKG reductase isadded exogenously to the bioreactor.

In another embodiment for making KLG, the bioreactor is charged withPantoea citrea cells comprising a modification in nucleic acid encodingan endogenous enzymatic activity which phosphorylates D-glucose at its6th carbon and/or in nucleic acid encoding an enzymatic activity thatphosphorylates D-gluconate at its 6th carbon and further comprisesnucleic acid encoding membrane-bound GDH, appropriate enzymes andcofactor, and D-gluconic acid is added which is converted to DKG. Thereaction mixture is then made anaerobic and glucose is added. The GDHconverts the glucose to GA, and the reductase converts DKG to KLG, whilecofactor is recycled. When these reactions are completed, oxygen isadded to convert GA to DKG, and the cycles continue.

In the in vitro biocatalytic process, the carbon source and metabolitesthereof proceed through enzymatic oxidation steps or enzymatic oxidationand enzymatic reducing steps which may take place outside of the hostcell intracellular environment and which exploit the enzymatic activityassociated with the host cell and proceed through a pathway to producethe desired ASA intermediate. The enzymatic steps may proceedsequentially or simultaneously within the bioreactor and some have aco-factor requirement in order to produce the desired ASA intermediate.The present invention encompasses an in vitro process wherein the hostcells are treated with an organic substance, such that the cells arenon-viable, yet enzymes remain available for oxidation and reduction ofthe desired carbon source and/or metabolites thereof in the biocatalysisof carbon source to ASA intermediate.

The bioreactor may be performed in a batch process or in a continuousprocess. In a batch system, regardless of what is added, all of thebroth is harvested at the same time. In a continuous system, the brothis regularly removed for downstream processing while fresh substrate isadded. The intermediates produced may be recovered from the fermentationbroth by a variety of methods including ion exchange resins, absorptionor ion retardation resins, activated carbon,concentration-crystallization, passage through a membrane, etc.

In some embodiments, the host cell is permeabilized or lyophilized(Izumi et al., J. Ferment. Technol. 61 (1983)135-142) as long as thenecessary enzymatic activities remain available to convert the carbonsource or derivatives thereof. The bioreactor may proceed with someenzymatic activities being provided exogenously and in an environmentwherein solvents or long polymers are provided which stabilize orincrease the enzymatic activities. In some embodiments, methanol orethanol is used to increase reductase activity. In another embodiment,Gafquat is used to stabilise the reductase (see Gibson et al., U.S. Pat.No. 5,240,843).

In some embodiments of the invention, a carbon source is converted toKLG in a process which involves co-factor regeneration. In thisenzymatic cofactor regeneration process, one equivalent of D-glucose isoxidized to one equivalent of D-gluconate, and one equivalent of NADP+is reduced to one equivalent of NADPH by the catalytic action of GDH.The one equivalent D-gluconate produced by the GDH is then oxidized toone equivalent of 2-KDG, and then to one equivalent of 2,5-DKG by theaction of membrane bound dehydrogenases GADH and KDGDH, respectively.The one equivalent 2,5-DKG produced is then reduced to one equivalent of2-KLG, and the NADPH is oxidized back to one equivalent of NADP+ by theaction of 2,5-DKG reductase, effectively recycling the equivalentcofactor to be available for a second equivalent of D-glucose oxidation.Other methods of cofactor regeneration can include chemical,photochemical, and electrochemical means, where the equivalent oxidizedNADP+ is directly reduced to one equivalent of NADPH by either chemical,photochemical, or electrochemical means.

C. Host Cells Producing ASA

Any productive or reducing enzymes necessary for directing a host cellcarbohydrate pathway into an ASA intermediate, such as, for example,KDG, DKG or KLG, can be introduced via recombinant DNA techniques knownto those of skill in the art if such enzymes are not naturally occurringin the host cell. Alternatively, enzymes that would hinder a desiredpathway can be inactivated by recombinant DNA methods. The presentinvention encompasses the recombinant introduction or inactivation ofany enzyme or intermediate necessary to achieve a desired pathway.

In some embodiments, Enterobacteriaceae strains that have been cured ofa cryptic plasmid are used in the production of ASA, see PCT TWO98/59054.

In some embodiments, the host cell used for the production of an ASAintermediate is Pantoea citrea, for example, ATCC accession number39140. Sources for nucleic acid encoding productive or reducing enzymeswhich can be used in the production of ASA intermediates in Pantoeaspecies include the following:

TABLE I ENZYME CITATION glucose dehydrogenase Smith et al. 1989,Biochem. J. 261:973; Neijssel et al. 1989, Antonie Van Leauvenhoek56(1):51–61 Cha, et al, Appl. Environ. Microbiol 63(1), 71–76 (1997);Pujol, C. J., et al, Microbiol. 145, 1217– 1226 gluconic aciddehydrogenase Matsushita et al. 1979, J. Biochem. 85:1173; Kulbe et al.1987, Ann. N.Y. Acad Sci 6:552 (Los Angeles) Pujol, C. J., et al, J. ofBacteriol 63(1), 71–76 (1999) Yum, D, et al, J. of Bacteriol183(8)2230–2237 2-keto-D-gluconic acid Stroshane 1977 Biotechnol. BioEngdehydrogenase 19(4) 459 2-keto gluconate reductase J. Gen. Microbiol.1991, 137:1479 Pujols, et al, J. of Bacterial. 182(8), (2000)2,5-diketo-D-gluconic acid U.S. Pat. Nos.: reductase 5,795,761;5,376,544; 5,583,025; 4,757,012; 4,758,514; 5,008,193; 5,004,690;5,032,514

D. Recovery of ASA Intermediates

Once produced, the ASA intermediates can be recovered and/or purified byany means known to those of skill in the art, including, lyophilization,crystallization, spray-drying, and electrodialysis, etc. Electrodialysismethods for purifying ASA and ASA intermediates such as KLG aredescribed in for example, U.S. Pat. No. 5,747,306 issued May 5, 1998 andU.S. Pat. No. 4,767,870, issued Aug. 30, 1998. Alternatively, theintermediates can also be formulated directly from the fermentationbroth or bioreactor and granulated or put in a liquid formulation.

KLG produced by a process of the present invention may be furtherconverted to ascorbic acid and the KDG to erythorbate by means known tothose of skill in the art, see for example, Reichstein and Grussner,Helv. Chim. Acta., 17, 311-328 (1934). Four stereoisomers of ascorbicacid are possible: L-ascorbic acid, D-araboascorbic acid (erythorbicacid), which shows vitamin C activity, L-araboascorbic acid, andD-xyloascorbic acid.

E. Assay Conditions

Methods for detection of ASA intermediates, ASA and ASA stereoisomersinclude the use of redox-titration with 2,6 dichloroindophenol (Burtonet al. 1979, J. Assoc. Pub. Analysts 17:105) or other suitable reagents;high-performance liquid chromatography (HPLC) using anion exchange (J.Chrom. 1980, 196:163); and electro-redox procedures (Pachia, 1976, Anal.Chem. 48:364). The skilled artisan will be well aware of controls to beapplied in utilizing these detection methods.

Fermentation media in the present invention must contain suitable carbonsubstrates which will include but are not limited to monosaccharidessuch as glucose, oligosaccharides such as lactose or sucrose,polysaccharides such as starch or cellulose and unpurified mixtures froma renewable feedstocks such as cheese whey permeate, comsteep liquor,sugar beet molasses, and barley malt. Additionally the carbon substratemay also be one-carbon substrates such as carbon. While it iscontemplated that the source of carbon utilized in the present inventionmay encompass a wide variety of carbon containing substrates and willonly be limited by the choice of organism, the preferred carbonsubstrates include glucose and/or fructose and mixtures thereof. Byusing mixtures of glucose and fructose in combination with the modifiedgenomes described elsewhere in this application, uncoupling of theoxidative pathways from the catabolic pathways allows the use of glucosefor improved yield and conversion to the desired ascorbic acidintermediate while utilizing the fructose to satisfy the metalbolicrequirements of the host cells.

Although it is contemplated that all of the above mentioned carbonsubstrates are suitable in the present invention preferred are thecarbohydrates glucose, fructose or sucrose. The concentration of thecarbon substrate is from about 55% to about 75% on a weight/weightbasis. Preferably, the concentration is from about 60 to about 70% on aweight/weight basis. The inventors most preferably used 60% or 67%glucose.

In addition to an appropriate carbon source, fermentation media mustcontain suitable minerals, salts, vitamins, cofactors and bufferssuitable for the growth or the cultures and promotion of the enzymaticpathway necessary for ascorbic acid intermediate production.

Culture Conditions:

Precultures:

Typically cell cultures are grown at 25 to 32° C., and preferably about28 or 29° C. in appropriate media. While the examples describe growthmedia used, other exemplary growth media useful in the present inventionare common commercially prepared media such as Luria Bertani (LB) broth,Sabouraud Dextrose (SD) broth or Yeast medium (YM) broth. Other definedor synthetic growth media may also be used and the appropriate mediumfor growth of the particular microorganism will be known by someoneskilled in the art of microbiology or fermentation science.

Suitable pH ranges preferred for the fermentation are between pH 5 to pH8 where pH 7 to pH 7.5 for the seed flasks and between pH 5 to pH 6 forthe reactor vessel.

It will be appreciated by one of skill in the art of fermentationmicrobiology that, now that Applicants have demonstrated the feasibilityof the process of the present invention a number of factors affectingthe fermentation processes may have to be optimized and controlled inorder to maximize the ascorbic acid intermediate production. Many ofthese factors such as pH, carbon source concentration, and dissolvedoxygen levels may affect the enzymatic process depending on the celltypes used for ascorbic acid intermediate production.

Batch and Continuous Fermentations:

The present process employs a fed-batch method of fermentation for itsculture systems. A classical batch fermentation is a closed system wherethe composition of the media is set at the beginning of the fermentationand not subject to artificial alterations during the fermentation. Thus,at the beginning of the fermentation the media is inoculated with thedesired organism or organisms and fermentation is permitted to occuradding nothing to the system. Typically, however, a “batch” fermentationis batch with respect to the addition of carbon source and attempts areoften made at controlling factors such as pH and oxygen concentration.In batch systems the metabolite and biomass compositions of the systemchange constantly up to the time the fermentation is stopped. Withinbatch cultures cells moderate through a static lag phase to a highgrowth log phase and finally to a stationary phase where growth rate isdiminished or halted. If untreated, cells in the stationary phase willeventually die. Cells in log phase generally are responsible for thebulk of production of end product or intermediate.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch fermentation processes are also suitable in the presentinvention and comprise a typical batch system with the exception thatthe substrate is added in increments as the fermentation progresses.Fed-Batch systems are useful when catabolite repression is apt toinhibit the metabolism of the cells and where it is desirable to havelimited amounts of substrate in the media. Measurement of the actualsubstrate concentration in Fed-Batch systems is difficult and istherefore estimated on the basis of the changes of measurable factorssuch as pH, dissolved oxygen and the partial pressure of waste gasessuch as CO₂. Batch and Fed-Batch fermentations are common and well knownin the art and examples may be found in Brock, supra.

Although the present invention is performed in batch mode it iscontemplated that the method would be adaptable to continuousfermentation methods. Continuous fermentation is an open system where adefined fermentation media is added continuously to a bioreactor and anequal amount of conditioned media is removed simultaneously forprocessing. Continuous fermentation generally maintains the cultures ata constant high density where cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or end product concentration.For example, one method will maintain a limiting nutrient such as thecarbon source or nitrogen level at a fixed rate and allow all otherparameters to moderate. In other systems a number of factors affectinggrowth can be altered continuously while the cell concentration,measured by media turbidity, is kept constant. Continuous systems striveto maintain steady state growth conditions and thus the cell loss due tomedia being drawn off must be balanced against the cell growth rate inthe fermentation. Methods of modulating nutrients and growth factors forcontinuous fermentation processes as well as techniques for maximizingthe rate of product formation are well known in the art of industrialmicrobiology and a variety of methods are detailed by Brock, supra.

It is contemplated that the present invention may be practiced usingeither batch, fed-batch or continuous processes and that any known modeof fermentation would be suitable. Additionally, it is contemplated thatcells may be immobilized on a substrate as whole cell catalysts andsubjected to fermentation conditions for ascorbic acid intermediateproduction.

Identification and Purification of Ascorbic Acid Intermediates:

Methods for the purification of the desired ascorbic acid intermediatefrom fermentation media are known in the art.

The specific ascorbic acid intermediate may be identified directly bysubmitting the media to high pressure liquid chromatography (HPLC)analysis. Preferred in the present invention is a method wherefermentation media is analyzed on an analytical ion exchange columnusing a mobile phase of 0.01N sulfuric acid in an isocratic fashion.

EXAMPLES General Methods

Materials and Methods suitable for the maintenance and growth ofbacterial cultures were found in Manual of Methods for GeneralBacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow,Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips,eds), pp. 210-213. American Society for Microbiology, Washington, D.C.or Thomas D. Brock in Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition (1989) Sinauer Associates, Inc.,Sunderland, Mass. All reagents and materials used for the growth, and ofbacterial cells were obtained from Diffco Laboratories (Detroit, Mich.),Aldrich Chemicals (Milwaukee, Wis.) or Sigma Chemical Company (St.Louis, Mo.) unless otherwise specified.

Growth medium for the precultures or inoculuum is commercially availableand preparations such as Luria Bertani (LB) broth, Sabouraud Dextrose(SD) broth or Yeast medium (YM) broth are obtainable from GIBCO/BRL(Gaithersburg, Md.). LB-50 amp is Luria-Bertani broth containing 50.mu.g/ml ampicillin.

Fermentation Media:

Two basic fermentation media were prepared for use in the followingexamples, and identified as Seed Flask Media and Fermentation Media.These basic media were modified by altering the carbon source or by theaddition of other reagents such as sulfite. The reagents useful for therespective media include KH₂PO₄, K₂HPO₄, MgSO4 7H₂O, Difco Soytone,Sodium citrate, Fructose, (NH₄)₂SO₄, Nicotinic acid, FeCl₃.6H₂O, andtrace salts, including, but not limited to ZnSO₄.7H₂O, MnSO₄.H₂O, andNa₂MoO₄.2H₂O); KH₂PO₄, MgSO4.7H2O, (NH₄)₂SO₄, Mono-sodium glutamate,ZnSO₄.7H₂O, MnSO₄.H₂O, Na₂MoO₄.2H₂O, FeCl₃.6H₂O, Choline chloride, MazuDF-204 (an antifoaming agent), Nicotinic acid, Ca-pantothenate and HFCS(42DE). HFCS can also be made according to the desired ratios of glucoseto fructose, e.g., a frucose/glucose solution made of 27.3 g/L powderedfructose, 25.0 g/L powdered glucose.Cells:

All commercially available cells used in the following examples wereobtained from the ATCC and are identified in the text by their ATCCnumber. Recombinant P. citrea cells (ATCC39140) were used as ascorbicacid intermediate producers and were constructed as described inExamples 4 and 5. Enzymatic assays and genome analysis revealed that thestrains MDP41 and DD6 lacked the genes encoding the glucokinase,gluconokinase and both enzymes whereas the wild-type strains containedgenes encoding the glucokinase and/or gluconokinase enzymes.

Ascorbic Acid Intermediate Analysis:

The presence of ascorbic acid intermediates, e.g., 2-KLG, was verifiedby running a HPLC analysis. Fermentation reactor vessel samples weredrawn off the tank and loaded onto Dionex (Sunnyvale, Calif., ProductNo. 043118) Ion Pac AS 10 column (4 mm times 250 mm) connected to aWaters 2690 Separation Module and a Waters 410 DifferentialRefractometer (Milford, Mass.).

Methods of Assaying for Production of Ascorbic Acid Intermediate

Methods for determining the yield, OUR, and CER were described earlierin the definition section.

Recombinant Methods

Vector Sequences

Expression vectors used the methods of the present invention comprise atleast one promoter associated with the enzyme, which promoter isfunctional in the host cell. In one embodiment of the present invention,the promoter is the wild-type promoter for the selected enzyme and inanother embodiment of the present invention, the promoter isheterologous to the enzyme, but still functional in the host cell. Inone embodiment of the present invention, nucleic acid encoding theenzyme is stably integrated into the microorganism genome.

In some embodiments, the expression vector contains a multiple cloningsite cassette which preferably comprises at least one restrictionendonuclease site unique to the vector, to facilitate ease of nucleicacid manipulation. In a preferred embodiment, the vector also comprisesone or more selectable markers. As used herein, the term selectablemarker refers to a gene capable of expression in the host microorganismwhich allows for ease of selection of those hosts containing the vector.Examples of such selectable markers include but are not limited toantibiotics, such as, erythromycin, actinomycin, chloramphenicol andtetracycline.

A preferred plasmid for the recombinant introduction of non-naturallyoccurring enzymes or intermediates into a strain of Enterobacteriaceaeis RSF1010, a mobilizable, but not self transmissible plasmid which hasthe capability to replicate in a broad range of bacterial hosts,including Gram− and Gram+ bacteria. (Frey et al., 1989, The Molecularbiology of IncQ plasmids. In: Thomas (Ed.), Promiscuous Plasmids of GramNegative Bacteria. Academic Press, London, pp. 79-94). Frey et al.(1992, Gene 113:101-106) report on three regions found to affect themobilization properties of RSF1010.

Transformation

General transformation procedures are taught in Current Protocols InMolecular Biology (vol. 1, edited by Ausubel et al., John Wiley & Sons,Inc. 1987, Chapter 9) and include calcium phosphate methods,transformation using DEAE-Dextran and electroporation. A variety oftransformation procedures are known by those of skill in the art forintroducing nucleic acid encoding a desired protein in a given hostcell. A variety of host cells can be used for recombinantly producingthe pathway enzymes to be added exogenously, including bacterial,fungal, mammalian, insect and plant cells.

In some embodiments of the process, the host cell is anEnterobacteriaceae. Included in the group of Enterobacteriaceae areErwinia, Enterobacter, Gluconobacter and Pantoea species. In the presentinvention, a preferred Enterobacteriaceae fermentation strain for theproduction of ASA intermediates is a Pantoea species and in particular,Pantoea citrea. In some embodiments, the host cell is Pantoea citreacomprising pathway enzymes capable of converting a carbon source to KLG.

Identification of Transformants

Whether a host cell has been transformed can be detected by thepresence/absence of marker gene expression which can suggest whether thenucleic acid of interest is present However, its expression should beconfirmed. For example, if the nucleic acid encoding a pathway enzyme isinserted within a marker gene sequence, recombinant cells containing theinsert can be identified by the absence of marker gene function.Alternatively, a marker gene can be placed in tandem with nucleic acidencoding the pathway enzyme under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the enzyme as well.

Alternatively, host cells which contain the coding sequence for apathway enzyme and express the enzyme may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridization and proteinbioassay or immunoassay techniques which include membrane-based,solution-based, or chip-based technologies for the detection and/orquantification of the nucleic acid or protein.

Additionally, the presence of the enzyme polynucleotide sequence in ahost microorganism can be detected by DNA-DNA or DNA-RNA hybridizationor amplification using probes, portions or fragments of the enzymepolynucleotide sequences.

The manner and method of carrying out the present invention may be morefully understood by those of skill in the art by reference to thefollowing examples, which examples are not intended in any manner tolimit the scope of the present invention or of the claims directedthereto. All references and patent publications referred to herein arehereby incorporated by reference.

EXAMPLES Example 1

Construction of a Genomic Library from P. citrea 139-2a

P.citrea genomic DNA was prepared using the DNA-Pure TM genomic DNAIsolation Kit (CPG, Lincoln Park, N.J.). 50 micrograms of this DNA waspartially digested with the restriction enzyme Sau3A accordingly themanufacturer recommendations (Roche Molecular Biochemicals,Indianapolis, Ind.). The products of the digestion were separated on a1% agarose gel and the DNA fragments of 3-5 kilobases were purified fromthe gel using the Qiaquick Gel extraction kit (Qiagen Inc. Valencia,Calif.). The resulting DNA was ligated with BamH1-linearized plasmidpBK-CMV (Stratagene, La Jolla, Calif.). A library of around 10××different plasmids was obtained in this way.

Example 2

Isolation of the Structural Gene for the Glucokinase Enzyme

To select for a plasmid that carries the glucokinase gene from P.citrea, the genomic library (see above) was transformed into a E. colistrain devoid of the glucokinase gene (glkA) and the PTS transportsystem, strain NF9, glk⁻, (Flores et al., Nat. Biotech. 14, 620-623).After transformation, the cells were selected for growth on M9 mediawith glucose as the only carbon source. With this strategy, plasmidsable to complement the glk⁻ or pts⁻ mutations were selected.

After 48 hrs. of incubation at 37° C., many colonies were visible.Several of these colonies were further purified and their plasmidsisolated and characterized by restriction analysis. It was found thatall the plasmids contained a common DNA fragment.

After re-transforming these plasmids back into NF9, glk⁻, all of themallowed growth on M9-glucose media, corroborating that they were able tocomplement at least one of the mutations present in NF9, glk⁻.

Plasmid pMD4 was isolated in this way and contains an insert of around3.9 kb. The insert in this plasmid was sequenced and it was found thatin a region of around 1010 bp, a gene with a strong similarity to the E.coli glkA gene was present. (SEQ ID 4.)

Example 3

Inactivation of the Glucokinase Gene by Homologous Recombination.

The general strategy to inactivate genes by homologous recombinationwith a a suicide vector has been delineated before (Miller andMekalanos., J. Bacteriol. 170 (1988) 2575-2583). To inactivate the glkgene from P. citrea by this approach two plasmids were constructed: pMD5and pMD6.

To construct pMD5, plasmid pMD4 was digested with the Ncol and SnaB1restriction enzymes accordingly manufacturer specifications. (RocheMolecular Biochemicals, Indianapolis, Ind.). The cohesive ends generatedby these enzymes were blunt-ended with T4 polymerase using standardtechniques. This DNA was ligated with a loxP-Cat-loxP cassette isolatedfrom pLoxCat2 as a Spel-EcoRV DNA fragment. (Palmeros et al., Gene(2000) 247, 255-264.). This cassette codes for Chloramphenicolresistance. The ligation mixture was transformed into TOPIO competentcell (Invitrogen, Carlsbard Calif.). selecting for growth onChloramphenicol 10 micrograms/ml. Several colonies were obtained after18 hr. incubation at 37° C. The plasmids of some of these colonies werepurified and characterized by restriction analysis. The presence of theloxP-Cat-loxP and the deletion of the DNA region between the Ncol andSnaB1 sites in the glk gene was confirmed. The plasmid with theseproperties was named pMD5.

To construct pMD6, plasmid pMD5 was digested with the BamH1 and Cel11restriction enzymes. The DNA fragment containing the glk geneinterrupted with the loxP-cassette was ligated to a EcoRV-Bsal DNAfragment isolated from plasmid pR6Kori1 (unpublished results). Thisfragment contains the R6K origin of replication and the Kanamycinresistance gene. The ligation mixture was transformed into strain SY327(Miller and Mekalanos., ibid.) and transformants were selected on platescontaining kanamycin and chloramphenicol (20 and 10 micrograms/mlrespectively). Several colonies were obtained after 24 hr. incubation at37° C. The plasmids of some of these colonies were purified andcharacterized by restriction analysis. The presence of the loxP-Cat-loxPand the R6K origin was confirmed. The plasmid with these characteristicswas named pMD6.

One characteristic of pMD6 and R6K derivatives in general, is that theycan only replicate in strains that carry the pir gene from plasmid R6K(Miller and Mekalanos., ibid.). P. citrea does not contain the pir geneor sustains replication of pMD6. After transforming pMD6 into P. citrea139-2a and selecting for Cm (R) strains, the proper gene replacement byhomologous recombination was obtained . The inactivation of theglucokinase gene was confirmed by assaying Glucokinase activity usingthe glucokinase-glucose-6-phosphate deydrogenase coupled assay describedby Fukuda et al., (Fukuda Y., Yamaguchi S., Shimosaka M., Murata K. andKimura A. J. Bacteriol. (1983) vol.156: pp. 922-925). The P. citreastrain where the glucokinase inactivation was confirmed was named MDP4.

Further confirmation of the inactivation of the glucokinase gene wasgenerated by comparing the size PCR products obtained using chromosomalDNA from 139-2a or MDP4 strains and primers that hybridize with theglucokinase structural gene (SEQ ID NO: 8 and 9, respectively). Withthis approach, the size of the PCR products should reflect that theloxP-Cat-loxP cassette was cloned in the glucokinase structural gene.

Example 4

Removal of the Chloramphenicol Resistance Marker in MDP4

After overnight growth on YENB medium (0.75% yeast extract, 0.8%nutrient broth) at 30° C., P. citrea MDP40 in a water suspension waselectrotransformed with plasmid pJW168 (. (Palmeros et al., Gene (2000)247, 255-264.). which contained the bacteriophage P1 Cre recombinasegene (IPTG-inducible), a temperature-sensitive pSC101 replicon, and anampicillin resistance gene. Upon outgrowth in SOC medium at 30° C.,transformants were selected at 30° C. (permissive temperature for pJW168replication) on LB agar medium supplemented with carbenicillin (200μg/ml) and IPTG (1 mM). Two serial overnight transfers of pooledcolonies were carried out at 35° C. on fresh LB agar medium supplementedwith carbenicillin and IPTG in order to allow excision of thechromosomal chloramphenicol resistance gene via recombination at theloxP sites mediated by the Cre recombinase (Hoess and Abremski, J. Mol.Biol., 181:351-362). Resultant colonies were replica-plated on to LBagar medium supplemented with carbenicillin and IPTG and LB agarsupplemented with chloramphenicol (12.5 μg/ml) to identify colonies at30° C. that were carbenicillin-resistant and chloramphenicol-sensitiveindicating marker gene removal. An overnight 30° C. culture of one suchcolony was used to inoculate 10 ml of LB medium. Upon growth at 30° C.to OD (600 nm) of 0.6, the culture was incubated at 35° C. overnight.Several dilutions were plated on prewarmed LB agar medium and the platesincubated overnight at 35° C. (the non-permissive temperature for pJW168replication). Resultant colonies were replica-plated on to LB agarmedium and LB agar medium supplemented with carbenicillin (200 μg/ml) toidentify colonies at 30° C. that were carbenicillin-sensitive,indicating loss of plasmid pJW168. One such glK mutant, MDP41, wasfurther analyzed by genomic PCR using primers SEQ ID NO:5 and SEQ IDNO:6 and yielded the expected PCR product (data not shown).

Example 5

Inactivation of the Gluconate Kinase Gene by Homologous Recombination.

The general strategy utilized to inactivate the gluconate kinase gene ofP. citrea is presented in FIG. 10, was in essence the same used toinactivate the glucokinase gene as described in example 3. Briefly,after isolating and sequencing a plasmid that allowed a E. coli straingntK⁻ idnK⁻, to grow using gluconate as the only carbon source (data notshown); a DNA fragment containing the structural gene for the gluconatekinase gene was generated by PGR using primers SEQ. ID NO: 7 and SEQ. IDNO: 8. This approximately 3 kb PGR product was cloned in a multicopyplasmid containing an R6K origin of replication. A unique Pstlrestriction site located in the gluconate kinase structural gene asshown in SEQ. ID NO: 2, was utilized to insert a loxP-Cat-loxP cassette.This construction was transferred to the chromosome of the P. citreastrain MDP41 by homologous recombination.

The correct interruption of the gluconate kinase with the loxP-Cat-loxPcassette was confirmed by PCR, using primers SEQ ID NO: 8 and SEQ ID NO:9.

The new strain, with both glucose and gluconate kinase inactivated wasnamed MDP5. This strain still contains the Cat marker inserted in thegluconate kinase structural gene. By repeating the procedure describedin example 4, a markerless strain was obtained and named DD6.

Experimental 6

The following illustrates the benefit of a double delete host cell(glucokinase and gluconokinase deleted Pantoea host cells) in terms ofO₂ demand.

Seed Train:

A vial of culture stored in liquid nitrogen is thawed in air and 0.75 mLis added to a sterile 2-L Erlenmeyer flasks containing 500 mL of seedmedium. Flasks are incubated at 29° C. and 250 rpm for 12 hours.Transfer criteria is an OD₅₅₀ greater than 2.5.

Seed Flask Medium

A medium composition was made according to the following:

Component Amount KH₂PO₄ 12.0 g/L K₂HPO₄ 4.0 g/L MgSO4•7H₂O 2.0 g/L DifcoSoytone 2.0 g/L Sodium citrate 0.1 g/L Fructose 5.0 g/L (NH₄)₂SO₄ 1.0g/L Nicotinic acid 0.02 g/L FeCl₃•6H₂O 5 mL/L (of a 0.4 g/L stocksolution) Trace salts 5 mL/L (of the following solution: 0.58 g/LZnSO₄•7H₂O, 0.34 g/L MnSO₄H₂O, 0.48 g/L Na₂MoO₄•2H₂O)

The pH of the medium solution was adjusted to 7.0±0.1 unit with 20%NaOH. Tetracycline HCI was added to a final concentration of 20 mg/L (2mL/L of a 10 g/L stock solution). The resulting medium solution was thenfilter sterilized with a 0.2μ filter unit. The medium was thenautoclaved and 500 mL of the previously autoclaved medium was added to2-L Erlenmeyer flasks.

Production Fermentor

Additions to the reactor vessel prior to sterilization

Component Conc KH₂PO₄ 3.5 g/L MgSO4•7H2O 1.0 g/L (NH₄)₂SO₄ 0.92 g/LMono-sodium glutamate 15.0 g/L ZnSO₄•7H₂O 5.79 mg/L MnSO₄•H₂O 3.44 mg/LNa₂MoO₄•2H₂O 4.70 mg/L FeCl₃•6H₂O 2.20 mg/L Choline chloride 0.112 g/LMazu DF-204 0.167 g/L

The above constituted media was sterilized at 121° C. for 45 minutes.

After tank sterilization, the following additions were made to thefermentation tank:

Component Conc Nicotinic acid 16.8 mg/L Ca-pantothenate 3.36 mg/L HFCS(42DE) 95.5 g/L (gluconate or glucose if desired as the particularstarting substrate)

The final volume after sterilization and addition of post-sterilizationcomponents was 6.0 L. The so prepared tank and medium were inoculatedwith the full entire contents from seed flask prepared as described togive a volume of 6.5 L.

Growth conditions were at 29° C. and pH 6.0. Agitation rate, backpressure, and air flow are adjusted as needed to keep dissolved oxygenabove zero.

Results

The oxidative pathway for ascorbic acid intermediates is depicted inFIG. 10. By determining the amount of carbon dioxide produce (CER), onecan calculate the amount of carbon utilized by the catabolic pathway andthus measure the uncoupling of the catabolic and productive (oxidative)pathways since the sole source of carbon for CO₂ is from the carbonsubstrate, no additional CO₂ having been supplied into the reactorvessel. When the wild-type organism was utilized in the fermentationprocess, 63% of the glucose was converted to an ascorbic acidintermediate, while 37% was converted, as measured by the CER, tocatabolic products (FIG. 12). In the second phase of the study, thenucleic acid encoding glucokinase expression was run under conditions ofthe wild-type. As shown in FIG. 13A, CO₂ evolution decreased to about18%, as measured by CER. Thus glucose catabolism was reduced, but notcompletely uncoupled. In an attempt to ascertain the source, i.e. thepathway wherein the carbon substrate was being diverted to the catabolicpathway, gluconic acid was provided as the sole carbon source. As shownin FIG. 13B in comparison with FIG. 13A, gluconic acid was catabolizedat about the same rate as if glucose had been the carbon substrate. (83%gluconate converted to ascorbic acid intermediate v. 17% of the gluconicacid converted to the catabolic pathway (as measured by CER). See alsoTable 2:

TABLE 2 Fraction of Glucose Fraction of Gluconate converted to convertedto strain Metabolism DKG Metabolism DKG Wild-type 0.37 0.63 — —Glucokinase 0.18 0.82 0.17 0.83 delete (glkA) Gluconokinase 0.24 0.760.02 0.98 delete (gntK)

A last phase of the study was provided by the examination of the OUR andCER of a host cell having the genomic encoding for glucokinase andgluconokinase deleted from the host cell genome. FIG. 14 depicts 3%glucose was converted to CO₂, wereas a control (wild-type) exhibited a43% glucose to CO₂ yield. As a result, it appears that the wild-typeexhibited a high catabolism of glucose by the catabolic pathway, whichresulted in reduced yield and a high oxygen requirement. However, a dualdeletion of glucokinase and gluconokinase essentially inactivatedcatabolism to less than 10 percent, less than 5 percent and particularly3 or less % of the initial carbon substrate.

Conclusions

The double mutant of glucokinase and gluconokinase appeared to shuntalmost all of the glucose to 2,5-DKG, about 98%.

Example 7

Production of Glycerol from Fructose.

To demonstrate that Pantoea citrea can be used to produce chemicalcompounds derived from fructose, glycerol was produced using theapproach described by Empatage et al., [Emptage,M., Haynie,S.,Laffend,L., Pucci,J. and Whited,G. Process for the biological productionof 1,3-propanediol with high titer. Patent: TWO 01 12833-A 4122-FEB-2001; E.I. DU PONT DE NEMOURS AND COMPANY; GENENCORINTERNATIONAL, INC.]. Briefly, this approach uses two enzymes from yeastto convert dihydroxyacetone phosphate (DHAP) into glycerol as shown inthe following reaction:

The genes for the GPDI and GPP2 enzymes were cloned in a multicopyplasmid pTrc99 under the control of the Trc promoter (Empatage et al.,2001). This plasmid (pAH48) is able to produce high levels of bothenzymes. The inventors recognized that to produce glycerol in P. citrea,it was desireable to eliminate or reduce the natural ability of thestrain to assimilate glycerol. A common glycerol catabolic pathway inmany bacteria, is through the action of the glycerol kinase [Lin E.C.Ann. Rev. Microbiol. 1976. 30:535-578. Glycerol dissimilation and itsregulation in bacteria]. The inventors found that the P. citrea was ableto grow in media containing glycerols as the only carbon source.Furthermore, inspection of the P. citrea genome sequence, showed that itpossesses a glycerol kinase gene, very similar to the glkA gene fromE.coli.

Thus, to eliminate the glycerol kinase activity, the structural gene ofthis enzyme (gene glpK) was inactivated. This was accomplished asdescribed in Examples 3 and 5 (inactivation of glucokinase andgluconokinase genes). Briefly, a 2.9 kb DNA fragment containing the glpkgene and flanking sequences, was obtained by PCR using chromosomal DNAfrom P. citrea and the primers disclosed in SEQ ID NO: 11 and SEQ ID NO:12. This 2.9 kb DNA fragment was cloned in a R6K vector as indicated inExamples 3 and 5. The DNA sequence of the glpK gene is shown in SEQ IDNO: 13, and the protein sequence of GlpK is shown in SEQ ID NO: 14.

Inspection of the glpk DNA sequence showed the presence of a Hpa1 site,which was chosen to insert the LoxP-Cat-LoxP cassette. Once the desiredplasmid construction was obtained, the glpK interruption was transferredto the chromosome of P. citrea strain 139-2a ps-, by homologousrecombination as described in example 3 and 5. The resulting P. citreaglpk:: Cm strain was named MDG1.

Once the interruption of the glpK gene in the P.citrea genome wasconfirmed, the effect of this mutation was evaluated. For such apurpose, strain MDG1 was grown in minimal media M9 containing glycerol0.4% as the only carbon source. After incubating the cells for 48 hoursat 30° C., no growth was observed, indicating that strain MDG1 lost theability to utilize glycerol as a carbon source.

Strain MDG1 was transformed with plasmid pAH48 (Emptage et al., 2001),and the resulting strain MDG2, was tested for its capacity to produceglycerol using fructose as the only carbon source. This was accomplishedby incubating the strain in minimal media containing 2% fructose as theonly carbon source. After incubating the cells for 24 hours at 30° C., asample was collected and analyzed by HPLC as described by Emptage et al.(2001). By doing this, it was found that strain MDG1 did not produce anyglycerol, while strain MDG2 produced 1.36 g/L of glycerol. These resultsdemonstrated that P. citrea was able to divert a substantial part offructose into the formation of glycerol.

Various other examples and modifications of the foregoing descriptionand examples will be apparent to a person skilled in the art afterreading the disclosure without departing from the spirit and scope ofthe invention, and it is intended that all such examples ormodifications be included within the scope of the appended claims. Allpublications and patents referenced herein are hereby incorporated byreference in their entirety.

1. A process for producing a product in a recombinant bacterial hostcell comprising, a) uncoupling an oxidative membrane bound productivepathway, wherein glucose is oxidized to gluconate from a catabolicmetabolic pathway selected from the group of glycolyals, pentose pathwayor both in a bacterial host cell, comprising inactivating by homologousrecombination a polynucleotide encoding a glucokinase having at least95% sequence identity to SEQ ID NO: 2, which phosphoryletes glucose asan initial carbon substrate an the catabolic pathway, b) culturing thebacterial host cell under conditions suitable for the production of aproduct in the presence of a carbon source comprising glucose, and c)obtaining the product, wherein the product is gluconic acid,2-keto-D-gluconate (2-KDG), 2,5-diketo-D-gluconate (2,5-DKG),2-keto-L-gulonic acid (2-KLG), erythorbic acid, 5-keto-D-gluconate(5-KDG), tartaric acid, or idonic acid.
 2. The process according toclaim 1, wherein the glucokinase has an amino acid sequence of SEQ IDNO.
 2. 3. The process according to claim 1, wherein inactivation is bydeletion of the polynucleotide encoding said glucokinase.
 4. The processaccording to claim 1, wherein the bacterial host cell is selected fromthe group consisting of Ewinia, Enterobacter, Corynabacteria,Acetobacter, Gluconobacter, Pentoea, Pseudomonas, Bacillus, andEschedchla cells.
 5. The process according to claim 1, wherein theproduct is gluconate, 2-KDG, 2,5-DKG or 2-KLG.
 6. The process accordingto claim 1 further comprising isolating the obtained product.
 7. Theprocess according to claim 1, wherein the uncoupled catabolic andproductive pathways function simultaneously end non-competitively in thebacterial host cell.
 8. The process according to claim 1, wherein therecombinant bacterial host cell is cultured by continuous cell culture.9. The process according to claim 1, wherein the recombinant bacterialhost cell is cultured by batch culture.
 10. The process according toclaim 4, wherein the bacterial host is a Pantoea cell.
 11. The processaccording to claim 10, wherein the Pantoea cell is a P. citrea cell. 12.The process according to claim 4, wherein the bacterial host cell is anErwinia, Enterobacter, Corynebacteria, Acetobacter or Gluconobactercell.
 13. The process according to claim 1 further comprisinginactivating a polynucleotide encoding a gluconokinase having at least95% sequence identity to the sequence of SEQ ID NO:
 4. 14. The processaccording to claim 13, wherein the gluconokinase has the sequence of SEQID NO:
 4. 15. A process for producing a product in a bacterial cellcomprising, a) obtaining a recombinant bacterial cell which has beenaltered by uncoupling an oxidative productive pathway, wherein glucoseis oxidized to gluconate from a catabolic metabolic pathway wherein thebacterial cell is selected from Erwinia, Enterobacter, Corynebacteria,Acetobacter, Bacillus, Gluconobacter, Pantoae, and Escherichla cells,said uncoupling comprising inactivating a polynucleotide encoding aglucokinase which phoaphorylates D-glucose at its 6^(th) carbon andhaving at least 95% sequence identity to SEQ ID NO: 2, b) culturing therecombinant bacterial cell under conditions suitable for the productionof a product in the presence of a carbon source comprising glucose, andc) producing the product, wherein the product is gluconuate,2-keto-D-gluconate, 2,5-diketo-D-gluconate, or 2-keto-L-guionic, andwherein the production of the product is enhanced compared to the levelof production of the same product in an unaltered bacterial cell underthe same conditions.
 16. The process according to claim 15, wherein theglucoklnase has at least 97% sequence identity with SEQ ID NO:
 2. 17.The process according to claim 15 further comprising recovering theproduct.
 18. The process according to claim 16, wherein the glucokinasehas at least 99% sequence identity with SEQ ID NO:
 2. 19. The processaccording to claim 15, further comprising inactivaing a gluconokinasehaving at least 95% sequence identity to SEQ ID NO:
 4. 20. The processaccording to claim 19, wherein the gluconokinase has at least 97%sequence identity to SEQ ID NO:
 4. 21. The process according to claim15, wherein the bacterial cell is a Pantoea cell.
 22. The processaccording to claim 20, wherein the Pantcea is P. citrea.
 23. The processaccording to claim 15, wherein the bacterial cell is E. coli.